Spared Nerve Injury Rats Research Articles

SNI and CFA induce similar changes in TRPV1 and P2X3 expressions in the acute phase but not in the chronic phase of pain.

Peripheral inflammation and nerve injury usually accompany each other. However, whether inflammatory and neuropathic pain share similar mechanisms at all stages is unknown. TRPV1 and P2X3 are two major ion channels in dorsal root ganglia (DRGs) and are involved in chronic pain. Here, their function and expression in DRGs at different phases of the two types of pain were investigated. Both the paw withdrawal threshold (PWT) and paw withdrawal latency were decreased in rats injected with complete Freud's adjuvant (CFA). However, only the PWT was decreased in rats with spared nerve injury (SNI). CFA increased the magnitude of the TRPV1-mediated Ca2+ response but not the P2X3-mediated Ca2+ response 14 days after injection. Consistent with this result, the P2X3 expression level in CFA rats was increased only at 3 days after injection. SNI surgery increased the magnitudes of the TRPV1- and P2X3-mediated Ca2+ responses and upregulated both TRPV1 and P2X3 expression in lumbar DRGs. The distributions of TRPV1 and P2X3 in DRGs after modeling were observed, and TRPV1 was found to be highly expressed mainly in the L4-L5 DRGs in CFA rats and in the L5-L6 DRGs in SNI rats. P2X3 was highly expressed in the L4-L6 DRGs in CFA rats 3 days after injection but was only highly expressed in the L4 DRG 14 days after modeling. On the other hand, SNI promoted the P2X3 expression L4-L5 DRGs 3 days after surgery, but only L6 DRG 14 days after modeling. All the results indicate that P2X3 and TPRV1 are involved in inflammatory and neuropathic pain by different expression levels and distributions in the lumbar DRG in the chronic stage.

Peripheral nerve injury promotes morphine-seeking behavior in rats during extinction

Chronic neuropathic pain and prescription opioid abuse represent highly interconnected societal problems. We used a rat model of spared nerve injury (SNI) and an intravenous drug self-administration paradigm to investigate the impact of a neuropathic pain state on morphine-seeking behavior in extinction (i.e. when morphine is withheld). SNI, sham-operated and naive groups exhibited similar levels of active lever presses for morphine infusions on a fixed ratio 1 (FR1) schedule. Self-administration of morphine, but not vehicle, attenuated nerve injury-induced mechanical allodynia in SNI rats. Under these same conditions, mechanical paw withdrawal thresholds in sham-operated and naive groups were largely unaltered. However, SNI rats showed higher levels of morphine-seeking behavior compared to sham-operated or naïve groups in extinction (i.e. when vehicle was substituted for morphine). Interestingly, the perseveration of morphine-seeking behavior observed during extinction was only present in the SNI group despite the fact that all groups had a similar history of morphine self-administration intake. Our results suggest that different motivational states associated with neuropathic pain promote morphine-seeking behavior in extinction. Drug self-administration paradigms may be useful for evaluating analgesic efficacy and motivational properties associated with opioid reinforcers in pathological pain states.

Elemene Emulsion Injection Administration Reduces Neuropathic Pain by Inhibiting Astrocytic NDRG2 Expression within Spinal Dorsal Horn.

To investigate the mechanisms underlying elemene-induced analgesia in rats with spared nerve injury (SNI). Sixty-five rats were equally divided into 5 groups using a random number table: naive group, sham group, SNI group, SNI + elemene (40 mg·kg-1·d-1) group and naive + elemene (40 mg·kg-1·d-1) group. An SNI rat model was established and the intervention were given respectively for 14 consecutive days. Von Frey filament tests and elevated plus-maze (EPM) tests were used to evaluate the effect of elemene on the mechanical threshold and anxiety, respectively. Immunoblotting and immunostaining were used to measure the expression of glial fibrillary acidic protein (GFAP) and NMYC downstream-regulated gene 2 (NDRG2) within the lumbar spinal dorsal horn (SDH). The SNI rat model exhibited a significant decrease in paw withdrawal threshold and exploratory behaviour in the EPM (P<0.05). Consecutive administration of elemene alleviated SNI-induced mechanical allodynia and anxiety in rats (P<0.05). Immunohistochemical data showed that elemene decreased SNI-induced upregulation of NDRG2 within the SDH (P<0.05). Double immunofluorescent staining data further showed that elemene decreased SNI-induced upregulation of the number of GFAP immunoreactive (-ir), NDRG-ir, and GFAP/NDRG2 double-labelled cells within the SDH (P<0.05). Immunoblotting data showed that elemene decreased SNI-induced upregulation of GFAP and NDRG2 within the SDH (P<0.05). Elemene possibly alleviated neuropathic pain by downregulating the expression of NDRG2 in spinal astrocytes in a rat model of SNI.

Anti-nociceptive effects of Sedum Lineare Thunb. on spared nerve injury-induced neuropathic pain by inhibiting TLR4/NF-κB signaling in the spinal cord in rats

Neuropathic pain is still a critical public health problem worldwide. Thereby, the search for novel and more effective strategies against neuropathic pain is urgently considered. It is known that neuroinflammation plays a crucial role in the pathogenesis of neuropathic pain. SedumLineare Thunb. (SLT), a kind of Chinese herb originated from the whole grass of Crassulaceae plant, was reported to possess anti-inflammatory activity. However, whether SLT has anti-nociceptive effect on neuropathic pain and its possible underlying mechanisms remains poorly elucidated. In this study, a rat model of neuropathic pain induced by spared nerve injury (SNI)was applied. SLT (p.o.) was administered to SNI rats once every day lasting for 14 days. Pain-related behaviors were assessed by using paw withdrawal threshold (PWT) and CatWalk gait parameters. Expression levels of inflammatory mediators and pain-related signaling molecules in the spinal cord were detected using western blotting assay. The results revealed that SLT (30, 100, and 300 mg/kg, p.o.) treatment for SNI rats ameliorated mechanical hypersensitivity in a dose-dependent manner. Application of SLT at the most effective dose of 100 mg/kg to SNI rats not only significantly blocked microglial activation, but also markedly reduced the protein levels of spinal HMGB1, TLR4, MyD88, TRAF6, IL-1β, IL-6, and TNF-α, along with an enhancement in gait parameters. Furthermore, SLT treatment dramatically inhibited the phosphorylation levels of both IKK and NF-κB p65 but obviously improved both IκB and IL-10 protein expression in the spinal cord of SNI rats. Altogether, these data suggested that SLT could suppress spinal TLR4/NF-κB signaling pathway in SNI rats, which might at least partly contribute to its anti-nociceptive action, indicating that SLT may serveas a potential therapeutic agent for neuropathic pain.

Resveratrol mediates mechanical allodynia through modulating inflammatory response via the TREM2-autophagy axis in SNI rat model

BackgroundNeuropathic pain (NeuP) is a chronic and challenging clinical problem, with little effective treatment. Resveratrol has shown neuroprotection by inhibiting inflammatory response in NeuP. Recently, the triggering receptor expressed on myeloid cells 2 (TREM2) expressed by microglia was identified as a critical factor of inflammation in nervous system diseases. In this study, we explored whether resveratrol could ameliorate neuroinflammation and produce anti-mechanical allodynia effects via regulating TREM2 in spared nerve injury rats, as well as investigated the underlying mechanisms.MethodsA spared nerve injury (SNI) rat model was performed to investigate whether resveratrol could exert anti-mechanical allodynia effects via inhibiting neuroinflammation. To evaluate the role of TREM2 in anti-neuroinflammatory function of resveratrol, lentivirus coding TREM2 was intrathecally injected into SNI rats to activate TREM2, and the pain behavior was detected by the von Frey test. Furthermore, 3-methyladenine (3-MA, an autophagy inhibitor) was applied to study the molecular mechanisms of resveratrol-mediated anti-neuroinflammation using Western blot, qPCR, and immunofluorescence.ResultsThe TREM2 expression and number of the microglial cells were significantly increased in the ipsilateral spinal dorsal horn after SNI. We found that intrathecal administration of resveratrol (300ug/day) alleviated mechanical allodynia; obviously enhanced autophagy; and markedly reduced the levels of interleukin-1β, interleukin-6, and tumor necrosis factor-α in the ipsilateral spinal dorsal horn after SNI. Moreover, the number of Iba-1+ microglial cells and TREM2 expression were downregulated after resveratrol treatment. Intrathecal administration of lentivirus coding TREM2 and/or 3-MA in those rats induced deficiencies in resveratrol-mediated anti-inflammation, leading to mechanical allodynia that could be rescued via administration of Res. Furthermore, 3-MA treatment contributed to TREM2-mediated mechanical allodynia.ConclusionsTaken together, these data reveal that resveratrol relieves neuropathic pain through suppressing microglia-mediated neuroinflammation via regulating the TREM2-autophagy axis in SNI rats.

A Neural Circuit from Thalamic Paraventricular Nucleus to Central Amygdala for the Facilitation of Neuropathic Pain.

As one of the thalamic midline nuclei, the thalamic paraventricular nucleus (PVT) is considered to be an important signal integration site for many descending and ascending pathways that modulate a variety of behaviors, including feeding, emotions, and drug-seeking. A recent study has demonstrated that the PVT is implicated in the acute visceral pain response, but it is unclear whether the PVT plays a critical role in the central processing of chronic pain. Here, we report that the neurons in the posterior portion of the PVT (pPVT) and their downstream pathway are involved in descending nociceptive facilitation regarding the development of neuropathic pain conditions in male rats. Lesions or inhibition of pPVT neurons alleviated mechanical allodynia induced by spared nerve injury (SNI). The excitability of pPVT-central amygdala (CeA) projection neurons was significantly increased in SNI rats. Importantly, selective optogenetic activation of the pPVT-CeA pathway induced obvious mechanical hypersensitivity in naive rats. In addition, we used rabies virus (RV)-based and cell-type-specific retrograde transsynaptic tracing techniques to define a novel neuronal circuit in which glutamatergic neurons in the vlPAG were the target of the pPVT-CeA descending facilitation pathway. Our data suggest that this pPVTGlu+-CeA-vlPAGGlu+ circuit mediates central mechanisms of descending pain facilitation underlying persistent pain conditions.SIGNIFICANCE STATEMENT Studies have shown that the interactions between the posterior portion of the thalamic paraventricular nucleus (pPVT) and central amygdala (CeA) play a critical role in pain-related emotional regulation. However, most reports have associated this circuit with fear and anxiety behaviors. Here, an integrative approach of behavioral tests, electrophysiology, and immunohistochemistry was used to advance the novel concept that the pPVT-CeA pathway activation facilitates neuropathic pain processing. Using rabies virus (RV)-based and cell-type-specific retrograde transsynaptic tracing techniques, we found that glutamatergic neurons in the vlPAG were the target of the pPVT-CeA pathway. Thus, this study indicates the involvement of a pPVTGlu+-CeA-vlPAGGlu+ pathway in a descending facilitatory mechanism underlying neuropathic pain.

Electroacupuncture Modulates Spinal BDNF/TrκB Signaling Pathway and Ameliorates the Sensitization of Dorsal Horn WDR Neurons in Spared Nerve Injury Rats.

Neuropathic pain is more complex and severely affects the quality of patients’ life. However, the therapeutic strategy for neuropathic pain in the clinic is still limited. Previously we have reported that electroacupuncture (EA) has an attenuating effect on neuropathic pain induced by spared nerve injury (SNI), but its potential mechanisms remain to be further elucidated. In this study, we designed to determine whether BDNF/TrκB signaling cascade in the spinal cord is involved in the inhibitory effect of 2 Hz EA on neuropathic pain in SNI rats. The paw withdrawal threshold (PWT) of rats was used to detect SNI-induced mechanical hypersensitivity. The expression of BDNF/TrκB cascade in the spinal cord was evaluated by qRT-PCR and Western blot assay. The C-fiber-evoked discharges of wide dynamic range (WDR) neurons in spinal dorsal horn were applied to indicate the noxious response of WDR neurons. The results showed that 2 Hz EA significantly down-regulated the levels of BDNF and TrκB mRNA and protein expression in the spinal cord of SNI rats, along with ameliorating mechanical hypersensitivity. In addition, intrathecal injection of 100 ng BDNF, not only inhibited the analgesic effect of 2 Hz EA on pain hypersensitivity, but also reversed the decrease of BDNF and TrκB expression induced by 2 Hz EA. Moreover, 2 Hz EA obviously reduced the increase of C-fiber-evoked discharges of dorsal horn WDR neurons by SNI, but exogenous BDNF (100 ng) effectively reversed the inhibitory effect of 2 Hz EA on SNI rats, resulting in a remarkable improvement of excitability of dorsal horn WDR neurons in SNI rats. Taken together, these data suggested that 2 Hz EA alleviates mechanical hypersensitivity by blocking the spinal BDNF/TrκB signaling pathway-mediated central sensitization in SNI rats. Therefore, targeting BDNF/TrκB cascade in the spinal cord may be a potential mechanism of EA against neuropathic pain.

Electroacupuncture ameliorates mechanical hypersensitivity by down-regulating spinal Janus kinase 2/signal transducer and activation of transcription 3 and interleukin 6 in rats with spared nerve injury.

Evidence shows that the Janus kinase 2 (JAK2)/signal transducer and activator of transcription 3 (STAT3) signaling pathway participates in the pathogenesis of neuropathic pain. Our previous study revealed that electroacupuncture (EA) attenuated neuropathic pain via activation of alpha-7 nicotinic acetylcholine receptor (α7nAChR) in the spinal cord. However, whether 2 Hz EA alleviates neuropathic pain by regulating the downstream molecules JAK2/STAT3 has not been fully clarified. Paw withdrawal threshold (PWT) was used as a marker of mechanical allodynia in rats with spared nerve injury (SNI). After applying 2 Hz EA on day 3, 7, 14 and 21 post-surgery, spinal expression of JAK2, STAT3 and pro-inflammatory cytokine interleukin (IL)-6 was examined using quantitative reverse transcription and real-time polymerase chain reaction (qRT-PCR) and Western blot analysis. Intrathecal injection of the α7nAChR antagonist alpha-bungarotoxin (α-Bgtx) was used to further explore the mechanism underlying the effects of 2 Hz EA on expression of JAK2/STAT3 in SNI rats. It was found that levels of spinal STAT3 and IL-6 mRNA, as well as levels of phosphorylated (p)-JAK2, p-STAT3 and IL-6 protein, were markedly increased in SNI rats. 2 Hz EA attenuated the SNI-induced up-regulation of p-JAK2, p-STAT3 and IL-6 expression in the spinal cord. Furthermore, intrathecal injection of α-Bgtx (1.0 μg/kg) not only inhibited the effect of 2 Hz EA on mechanical hypersensitivity but also ameliorated the down-regulation of p-JAK2, p-STAT3 and IL-6 expression induced by 2 Hz EA. This study revealed that 2 Hz EA attenuated SNI-induced mechanical hypersensitivity and the concomitant up-regulation of spinal JAK2, STAT3 and IL-6 in SNI rats, suggesting that suppression of the JAK2/STAT3 signaling pathway might be the mechanism underlying the therapeutic effect of 2 Hz EA on neuropathic pain.

Downregulation of microRNA‑29c reduces pain after child delivery by activating the oxytocin‑GABA pathway.

A significant decrease in the expression of spinal microRNA-29c (miR-29c), which is responsible for the regulation of oxytocin receptor (OXTR) expression, was observed in nerve injury pain during childbirth. The present study investigates whether spinal miR-29c could be a potential target for the treatment of pain, via the oxytocin (OT)-γ-aminobutyric acid (GABA) pathway. A spared nerve injury (SNI) rat model was established to induce neuropathic pain, simulating hyperalgesia. Spinal neurons were treated with OT to mimic the hormonal changes in the central nervous system after delivery. A change in the neuronal miniature inhibitory postsynaptic currents (mIPSCs) was observed in neurons, following the silencing of miR-29c or OT treatment with or without OXTR antagonist. The Von-Frey apparatus was used to measure the animal behaviors. Molecular biological experiments and electrophysical recordings in vivo and in vitro were performed to reveal the potential analgesic mechanisms. miR-29c was significantly downregulated (more than 8-fold) in the spinal dorsal horn of delivery+SNI rats compared with the SNI rats. The silencing of miR-29c resulted in increased pain threshold in SNI rats. Bioinformatics analysis indicated that OXTR was a potential target gene of miR-29c. The delivery+SNI rats presented with higher levels of OT in the cerebrospinal fluid compared with SNI rats, which indicated that the OT signaling pathway may participate in pain relief response. The increased expression of OXTR and GABA in delivery+SNI rats were observed in the miR-29c-silenced SNI rat model, suggesting that the silencing of miR-29c can mediate pain relief by enhancing the OT-GABA pathway. In addition, an electrophysiology assay was performed to assess the mIPSCs in neurons. The silencing of miR-29c in neurons increased the frequency and amplitude of mIPSCs but there was no influence on the decay time, which suggested that the spinal inhibitory neurons became more active, subsequently reducing the feeling of pain. The inhibition of OXTR reversed the enhanced inhibitory postsynaptic currents, indicating a crucial role for OXTR in the miR-29c-associated pain regulation. Taken together, the results of the present study suggested that spinal oxytocinergic inhibitory control plays an important role in pain relief in the neuropathic pain rat model undergoing vaginal delivery. Silencing spinal miR-29c may be a potential target for pain relief through the OT-GABA pathway.

AAV-Mediated Combination Gene Therapy for Neuropathic Pain: GAD65, GDNF, and IL-10

Neuropathic pain is a chronic pain state characterized by nerve damage, inflammation, and nociceptive neuron hyperactivity. As the underlying pathophysiology is complex, a more effective therapy for neuropathic pain would be one that targets multiple elements. Here, we generated recombinant adeno-associated viruses (AAVs) encoding three therapeutic genes, namely, glutamate decarboxylase 65, glial cell-derived neurotrophic factor, and interleukin-10, with various combinations. The efficacy for pain relief was evaluated in a rat spared nerve injury model of neuropathic pain. The maximal analgesic effect was achieved when the AAVs expressing all three genes were administered to rats with neuropathic pain. The combination of two virus constructs expressing the three genes was named KLS-2031 and evaluated as a potential novel therapeutic for neuropathic pain. Single transforaminal epidural injections of KLS-2031 into the intervertebral foramen to target the appropriate dorsal root ganglion produced notable long-term analgesic effects in female and male rats. Furthermore, KLS-2031 mitigated the neuroinflammation, neuronal cell death, and dorsal root ganglion hyperexcitability induced by the spared nerve injury. These results suggest that KLS-2031 represents a promising therapeutic option for refractory neuropathic pain.

Network Analysis of miRNA and mRNA Changes in the Prelimbic Cortex of Rats With Chronic Neuropathic Pain: Pointing to Inflammation.

Neuropathic pain (NP) is a complex, chronic pain condition caused by injury or dysfunction affecting the somatosensory nervous system. This study aimed to identify crucial mRNAs and microRNAs (miRNAs) in the prelimbic cortex (PL) of NP rats. mRNA and miRNA microarrays were applied in the present study. The miRNA-mRNA regulatory network was constructed by using ingenuity pathway analysis (IPA). A total of 35 differentially expressed (DE) RNAs (24 miRNAs and 10 mRNAs) were identified in the spared nerve injury (SNI) group compared with the control group. The DE miRNA-mRNA network showed that IL-6 and tumor necrosis factor (TNF) were core components. Mir-30c-5p and mir-16-5p were the most connected miRNAs in the network. Interestingly, four mRNAs (Rnase 4, Egr2, Rexo4, and Klf2) with significantly increased expression were abundantly expressed in microglia, which was verified by the real-time quantitative polymerase chain reaction (qPCR). Furthermore, the expression of Rnase4 and Egr2 decreased in M1-polarized macrophages and increased in M2-polarized macrophages. In conclusion, we screened dozens of DE mRNAs and miRNAs in the PL of SNI rats. The core of the DE mRNA and miRNA network pointed to molecules associated with inflammation. Four mRNAs (Rnase4, Egr2, Rexo4, and Klf2) might be the potential markers of M2 polarization.

Identification of the Hub Genes Related to Nerve Injury-Induced Neuropathic Pain

BackgroundThe reactivity enhancement of pain sensitive neurons in the nervous system is a feature of the pathogenesis for neuropathic pain (NP), yet the underlying mechanisms need to be fully understood. In this study, we made an attempt to clarify the NP-related hub genes and signaling pathways so as to provide effective diagnostic and therapeutic methods toward NP.MethodsMicroarray expression profile GSE30691 including the mRNA-seq data of the spared nerve injury (SNI)-induced NP rats was accessed from the GEO database. Then, genes associated with NP development were screened using differential analysis along with random walk with restart (RWR). GO annotation and KEGG pathway analyses were performed to explore the biological functions and signaling pathways where the genes were activated. Afterward, protein-protein interaction (PPI) analysis and GO analysis were conducted to further identify the hub genes which showed an intimate correlation with NP development.ResultsTotally 94 genes associated with NP development were screened by differential analysis and RWR analysis, and they were observed to be predominantly enriched in hormone secretion and transport, cAMP signaling pathway and other NP occurrence associated functions and pathways. Thereafter, the 94 genes were subjected to PPI analysis to find the genes much more associated with NP and a functional module composed of 48 genes were obtained. 8 hub genes including C3, C1qb, Ccl2, Cxcl13, Timp1, Fcgr2b, Gal, and Lyz2 were eventually identified after further association and functional enrichment analyses, and the expression of these 8 genes were all higher in SNI rats by comparison with those in Sham rats.ConclusionBased on the data collected from GEO database, this study discovered 8 hub genes that were closely related to NP occurrence and development, which help to provide potent theoretical basis for NP treatment.

Inhibition of P2X7R in the amygdala ameliorates symptoms of neuropathic pain after spared nerve injury in rats

The amygdala circuitry and P2X7 receptor (P2X7R) have both been shown to play important roles in the modulation of neuropathic pain (NP). However, little is known about the functional role of P2X7R in the amygdala for the regulation of NP. This study aims to evaluate the alleviative effect of intra-amygdala microinfusion of a pharmacological antagonist of P2X7R (A-438079) on NP and explore its possible mechanism of action. Male Sprague-Dawley rats were used to construct the animal model of NP through spared nerve injury (SNI). The SNI rats randomly received chronic bilateral microinjection of A-438079 (100 pmol/side) or saline into the amygdalae via cannulas. Mechanical paw withdrawal threshold (MWT) and thermal withdrawal duration (TWD) were measured by von Frey monofilaments. Besides, tail suspension test (TST), forced swimming test (FST), open field test (OFT) and sucrose preference test (SPT) were performed to assess depression- and anxiety-like behaviors. Immunofluorescence assay was employed to determine the levels of glial fibrillary acidic protein (GFAP), ionized calcium binding adaptor molecule 1 (IBA-1) and connexin 43 (Cx43) in the spinal cord. In addition, the change of growth associated protein 43 (GAP43) level in the spinal cord was assessed by Western blot. Our data showed that chronic treatment with A-438079 increased MWT and decreased TWD on days 11–21 post-SNI while decreased depression-like and anxiety-like behaviors. A-438079 administration significantly attenuated the elevated immunoreactivities of IBA-1 and GFAP in microglia and astrocytes after SNI. Furthermore, the decreased expression of GAP-43 in the spinal cord due to SNI was significantly attenuated by A-438079. However, when A-438079 and a pharmacological agonist (BzATP) of P2X7R were given simultaneously, all the effects caused by A-438079 alone were reversed. In brief, our study revealed the protective role of inhibiting P2X7R in the amygdala against symptoms associated with NP, possibly attributing to its inhibitory effects on spinal microglia and astrocytes.

Lidocaine Alleviates Neuropathic Pain and Neuroinflammation by Inhibiting HMGB1 Expression to Mediate MIP-1α/CCR1 Pathway.

High mobility group box1 (HMGB1) released from sensory nerve tissues can induce neuropathic pain. Whether HMGB1 is implicated in the mechanism underlying the effect of lidocaine in pain management remains to be determined. This study aims to explore the effect of lidocaine in a rat model of spared nerve injury (SNI) and the underlying mechanism. An SNI model was established via nerve ligation. Two weeks after the SNI model was established, rats were intrathecally injected with lidocaine, an HMGB1 antibody (HMG Ab), an MIP-1α antibody (MIP-1α Ab), a CCR1 inhibitor (CCR1-RS) or a CCR5 antagonist (CCR5-Mar). Pain behaviors were assessed before and after model establishment to calculate the number of spontaneous flinches (NSF), paw withdrawal threshold (PWT), paw withdrawal thermal latency (PWL) and sciatic function index (SFI). Cell apoptosis and the inflammatory response in the cerebrospinal fluid (CSF) were detected by TUNEL staining and ELISA. The mRNA and protein expression levels of MIP-1α, CCR1 and CCR5 were determined by RT-PCR and Western blotting. The expression levels of HMGB1, MIP-1α, CCR1 and CCR5 were measured by Western blotting and immunofluorescence. Pain behavior testing in SNI rats showed that SNI rats exhibited an increased NSF and adecreased PWT, PWL and SFI. Cell apoptosis in the spinal dorsal horn and the generation of inflammatory cytokines were enhanced in SNI rats, and the expression levelsof HMGB1, MIP-1α, CCR1 and CCR5 were upregulated. HMGB1 cytoplasmic translocation, the coexpression of MIP-1α with NeuN, and the coexpression of CCR1 and CCR5 with OX42 were also observed in SNI rats. Neuropathic pain and neuroinflammation were suppressed by the intrathecal injection of lidocaine, HMG Ab, MIP-1α Ab, CCR1-RS or CCR5-Mar. Lidocaine inhibited the expression levels of HMGB1, MIP-1α, CCR1 and CCR5, and the HMGB1 antibody suppressed the expression of MIP-1α, CCR1 and CCR5. Lidocaine attenuates neuropathic pain and neuroinflammation by inhibiting HMGB1 to regulate the MIP-1α/CCR1/CCR5 pathway. Graphical Abstract.

Electroacupuncture Treatment Suppresses Transcription Factor IRF8 in Spinal Cord of Rats with Spared Nerve Injury.

Objective Neuropathic pain with complex mechanisms has become a major public health problem that greatly impacts patients' quality of life. Therefore, novel and more effective strategies against neuropathic pain need further investigation. Electroacupuncture (EA) has an ameliorating effect on neuropathic pain following spared nerve injury (SNI), but the underlying mechanism remains to be fully clarified. Interferon regulatory factor 8 (IRF8), a critical transcription factor, was reported to be involved in the modulation of neuropathic pain. Here, we focused on exploring whether 2 Hz EA stimulation exerts an inhibitory action on spinal IRF8 in SNI rats. Methods In this study, SNI rats were treated with 2 Hz EA once every other day for 21 days. Paw withdrawal threshold (PWT) was applied to determine the analgesic effect of 2 Hz EA on SNI rats. The spinal IRF8 and CX3CRl expressions were detected with qRT-PCR and western blot, and immunofluorescence staining was used to evaluate colocation of IRF8 or CX3CRl with microglial activation marker CD11b in the spinal cord. Results It was found that SNI induced significant elevation of spinal IRF8 and CX3CRl mRNA and protein expression. Additionally, immunofluorescence results showed that SNI elicited the coexpression of IRF8 with CD11b, as well as CX3CRl with CD11b in the spinal cord. Meanwhile, 2 Hz EA treatment of SNI rats not only reduced IRF8 and CX3CRl mRNA and protein expression, but also reversed the coexpression of IRF8 or CX3CRl with CD11b in the spinal cord, along with an attenuation of SNI-evoked mechanical hypersensitivity. Conclusion This experiment highlighted that 2 Hz EA can inhibit IRF8 expression and microglial activation in the spinal cord of SNI rats. Hence, targeting IRF8 may be a promising therapeutic strategy for 2 Hz EA treatment of neuropathic pain.

A novel and potent neuronal Kv7 channel opener SCR2682 alleviates chronic pain in rats

Activation of neuronal Kv7/KCNQ/M‐current emerges as an effective intervention for treatment of hyperexcitability‐related neurological diseases including chronic pain that is prevalent and unmet medical need. The aim of this study was to evaluate the anti‐nociceptive effects of a novel and potent Kv7/KCNQ/M channel opener SCR2682 on chronic pain in rats. The pharmacological effects of SCR2682 on native M current and firing of dorsal root ganglia (DRG) neurons were examined using electrophysiological recordings. Two in vivo rat models of complete Freund’s adjuvant (CFA)‐induced inflammatory pain and spared nerve injury (SNI)‐induced neuropathic pain were used to evaluate anti‐nociceptive effects of SCR2682 by measuring the paw withdrawal mechanical threshold (PWMT) and/or thermal latency (PWTL). Locomotor activity in normal rats was tested for safety evaluation. The expression of KCNQ2 in L4 and L5 DRGs of rat SNI model was detected by quantitative RT‐PCR and western blot.We found that SCR2682 (0.1 μM) or an FDA approved anti‐epileptic drug retigabine (RTG) (10 μM) increased M current in rat DRG neurons approximately 30%, which was blocked by Kv7 channel inhibitor XE991 (3 μM). SCR2682 caused the resting membrane potential (RMP) hyperpolarization to −66.4 ± 3.7 mV from −56.7 ± 2.8 mV. Current clamp recordings revealed that perfusion of SCR2682 abolished the firing spikes of DRG neurons. In rat model of CFA‐induced inflammatory pain, intraperitoneal (i.p.) administration of SCR2682 at doses of 0.5, 1 and 2 mg/kg increased the PWMT and PWTL in a dose‐dependent manner, as compared with RTG (7 mg/kg) that also increased the PWMT and PWTL. In the model of SNI‐induced neuropathic pain, SCR2682 (0.5, 1 and 2 mg/kg) or RTG (7 mg/kg) increased the PWMT, and the anti‐nociceptive effect of SCR2682 (2 mg/kg) could be reversed by the channel specific blocker XE991 (3 mg/kg, i.p.). Furthermore, SCR2682 (2 mg/kg.) or RTG (7 mg/kg) significantly increased the expression of KCNQ2 mRNA and protein in DRG of SNI rats, and co‐injection of XE991 (3 mg/kg) with SCR2682 reduced the expression levels of KCNQ2. In the open field test, there was no significant difference in the travel total distance and mean speed of rats treated with RTG (7 mg/kg) or SCR2682 (0.5, 1 and 2 mg/kg). Taken together, our data show that the novel compound SCR2682 reduces the excitability of DRG sensory neurons by activating Kv7 channel, and intraperitoneal administration of SCR2682 alleviates chronic pain in rats.Support or Funding InformationThis project was supported by grants awarded to K.W. from National Natural Sciences Foundation of China (81573410) and the Ministry of Science and Technology of China (2018ZX09711001‐004‐006) and Science and Technology Program of Guangdong (2018B030334001).

Protein Kinase A Is Involved in Neuropathic Pain by Activating the p38MAPK Pathway to Mediate Spinal Cord Cell Apoptosis.

Neuropathic pain is a serious clinical problem to be solved. This study is aimed at investigating protein kinase A (PKA) expression in neuropathic pain and its possible mechanisms of involvement. A neuropathic pain-related gene expression dataset was downloaded from Gene Expression Omnibus, and differentially expressed genes were screened using the R software. cytoHubba was used to screen for hub genes. A spared nerve injury (SNI) rat model was established, and the paw withdrawal threshold was determined using von Frey filaments. Western blotting and immunofluorescence were used to detect the expression and cellular localization, respectively, of key proteins in the spinal cord. Western blot, ELISA, and TUNEL assays were used to detect cell signal transduction, inflammation, and apoptosis, respectively. Pka was identified as a key gene involved in neuropathic pain. After SNI, mechanical allodynia occurred, PKA expression in the spinal cord increased, the p38MAPK pathway was activated, and spinal cord inflammation and apoptosis occurred in rats. PKA colocalized with neurons, astrocytes, and microglia, and apoptotic cells were mainly neurons. Intrathecal injection of a PKA inhibitor not only relieved mechanical hyperalgesia, inflammatory reaction, and apoptosis in SNI rats but also inhibited p38MAPK pathway activation. However, intrathecal injection of a p38MAPK inhibitor attenuated mechanical hyperalgesia, inflammation, and apoptosis, but did not affect PKA expression. In conclusion, PKA is involved in neuropathic pain by activating the p38MAPK pathway to mediate spinal cord cell apoptosis.

Reduction of Silent Information Regulator 1 Activates Interleukin-33/ST2 Signaling and Contributes to Neuropathic Pain Induced by Spared Nerve Injury in Rats.

Emerging studies have demonstrated that interleukin (IL)-33 and its receptor ST2 act as key factors in inflammatory diseases. Moreover, accumulating evidence has suggested that cytokines, including tumor necrosis factor (TNF)-α and IL-1β, trigger an inflammatory cascade. SIRT1 has been shown to suppress the expression of inflammatory cytokines. However, the effects of SIRT1 on IL-33/ST2 signaling and initiation of the inflammatory cascade via modulation of TNF-α and IL-1β by IL-33 remain unclear. In the present study, we found that the dorsal root ganglion (DRG) IL-33 and ST2 were upregulated in a rat model of spared nerve injury (SNI) and intrathecal injection of either IL-33 or ST2 antibodies alleviated mechanical allodynia and downregulated TNF-α and IL-1β induced by SNI. In addition, activation of SIRT1 decreased enhanced DRG IL-33/ST2 signaling in SNI rats. Artificial inactivation of SIRT1 via intrathecal injection of an SIRT1 antagonist could induce mechanical allodynia and upregulate IL-33 and ST2. These results demonstrated that reduction in SIRT1 could induce upregulation of DRG IL-33 and ST2 and contribute to mechanical allodynia induced by SNI in rats.

Synaptotagmin 1 Is Involved in Neuropathic Pain and Electroacupuncture-Mediated Analgesic Effect.

Numerous studies have verified that electroacupuncture (EA) can relieve neuropathic pain through a variety of mechanisms. Synaptotagmin 1 (Syt-1), a synaptic vesicle protein for regulating exocytosis of neurotransmitters, was found to be affected by EA stimulation. However, the roles of Syt-1 in neuropathic pain and EA-induced analgesic effect remain unclear. Here, the effect of Syt-1 on nociception was assessed through an antibody blockade, siRNA silencing, and lentivirus-mediated overexpression of spinal Syt-1 in rats with spared nerve injury (SNI). EA was used for stimulating bilateral “Sanjinjiao” and “Zusanli” acupoints of the SNI rats to evaluate its effect on nociceptive thresholds and spinal Syt-1 expression. The mechanically and thermally nociceptive behaviors were assessed with paw withdrawal threshold (PWT) and paw withdrawal latency (PWL) at different temperatures, respectively, at day 0, 7, 8, 14, and 20. Syt-1 mRNA and protein levels were determined with qRT-PCR and Western blot, respectively, and its distribution was observed with the immunohistochemistry method. The results demonstrated Syt-1 antibody blockade and siRNA silencing increased ipsilateral PWTs and PWLs of SNI rats, while Syt-1 overexpression decreased ipsilateral PWTs and PWLs of rats. EA significantly attenuated nociceptive behaviors and down-regulated spinal Syt-1 protein levels (especially in laminae I-II), which were reversed by Syt-1 overexpression. Our findings firstly indicate that Syt-1 is involved in the development of neuropathic pain and that EA attenuates neuropathic pain, probably through suppressing Syt-1 protein expression in the spinal cord.

Electroacupuncture relieved neuropathic pain by suppressing L-Arg/NO/cGMP pathway in the lumbar spinal cord in rats with spared nerve injury

To observe the effect of electroacupuncture (EA) on changes of expression of L-Arg transporter 2 (CAT-2) mRNA and nitric oxide synthase (iNOS) mRNA and protein and contents of NO and cGMP of L4-L6 segments of spinal cord in rats with spared nerve injury (SNI), so as to reveal its mechanism underlying reducing neuropathic pain. A total of 120 male SD rats were randomly divided into sham operation, model, EA and NOS inhibitor (N omega-Nitro-L-arginine methyl ester hydrochloride, L-NAME) groups, with 30 rats in each group. The neuropathic pain model was established by ligating and cutting the tibial nerve and the common peroneal nerve. EA (2 Hz, 1-3 mA) was applied to "Weizhong" (BL40) and "Huantiao" (GB30)on the damaged hindlimb for 30 min, once daily from day 11 to 17 after SNI. Rats of the L-NAME group received i.p. of L-NAME (60 mg·kg-1·d-1) for 7 consecutive days. The mechanical pain threshold (PT) was determined before and 10 and 16 d after SNI, respectively. The expression le-vels of CAT-2 mRNA and iNOS mRNA, and iNOS protein in the L4-L6 segments of the spinal cord were detected by using reverse transcription - polymerase chain reaction (RT-PCR) and Western blot, respectively, and the contents of NO and cGMP of L4-L6 assayed using nitrate/nitrite reductase method and radioimmunoassay, respectively. After modeling, the PT was significantly decreased on day 10 and 16 after SNI in comparison with the sham operation group and their own baseline data of pre-operation in each group (P<0.01), and remarkably increased in the EA and L-NAME groups relevant to the model group on day 16 (P<0.01, P<0.05). Compared with the sham operation group, the expression levels of CAT-2 mRNA and iNOS mRNA and protein, as well as the contents of NO2－/NO3－and cGMP were signi-ficantly up-regulated in the model group (P<0.05, P<0.01). Following EA intervention, the levels of CAT-2 mRNA and iNOS mRNA and iNOS protein, and NO2－/NO3－and cGMP contents were all reversed in both EA and L-NAME groups (P<0.05, P<0.01). The effect of EA was significantly superior to that of L-NAME in raising the PT on day 16 after SNI (P<0.05), but obviously inferior to that of L-NAME in down-regulating the expression of CAT-2 mRNA and iNOS mRNA and protein (P<0.05). No significant differences were found between the EA and L-NAME groups in down-regulating NO2－/NO3－ andcGMP contents (P>0.05). EA intervention can effectively relieve neuropathic pain in SNI rats, which may be closely related to its function in suppressing L-Arg/NO/cGMP pathway in the lumbar spinal cord.