Rat Spinal Nerve Ligation Model Research Articles

Intrathecal gastrodin alleviates allodynia in a rat spinal nerve ligation model through NLRP3 inflammasome inhibition

BackgroundGastrodin (GAS), a main bioactive component of the herbal plant, Gastrodia elata Blume, has shown to have beneficial effects on neuroinflammatory diseases such as Alzheimer’s disease in animal studies and migraine in clinical studies. Inflammasome is a multimeric protein complex having a core of pattern recognition receptor and has been implicated in the development of neuroinflammatory diseases. Gastrodin has shown to modulate the activation of nucleotide-binding oligomerization domain (NOD)-like receptor protein 3 (NLRP3) inflammasome. This study investigated the effects of GAS on the intensity of mechanical allodynia and associated changes in NLRP3 inflammasome expression at the spinal level using L5/6 spinal nerve ligation model (SNL) in rats.MethodsIntrathecal (IT) catheter implantation and SNL were used for drug administration and pain model in male Sprague-Dawley rats. The effect of gastrodin or MCC950 (NLRP3 inflammasome inhibitor) on mechanical allodynia was measured by von Frey test. Changes in NLRP3 inflammasome components and interleukin-1β (IL-1β) and cellular expression were examined in the spinal cord and dorsal root ganglion.ResultsThe expression of NLRP3 inflammasome components was found mostly in the neurons in the spinal cord and dorsal root ganglion. The protein and mRNA levels of NLRP3, apoptosis-associated speck-like protein containing a caspase recruitment domain (ASC), caspase-1, and IL-1β were upregulated in SNL animals compared to Sham animals. IT administration of GAS significantly attenuated the expression of NLRP3 inflammasome and the intensity of SNL-induced mechanical allodynia. NLRP3 inflammasome inhibitor, MCC950, also attenuated the intensity of allodynia, but the effect is less strong and shorter than that of GAS.ConclusionsExpression of NLRP3 inflammasome and IL-1β is greatly increased and mostly found in the neurons at the spinal level in SNL model, and IT gastrodin exerts a significant anti-allodynic effect in SNL model partly through suppressing the expression of NLRP3 inflammasome.

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

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

A novel role for microtubule affinity-regulating kinases in neuropathic pain.

Neuropathic pain affects millions of patients, but there are currently few viable therapeutic options available. Microtubule affinity-regulating kinases (MARKs) regulate the dynamics of microtubules and participate in synaptic remodelling. It is unclear whether these changes are involved in the central sensitization of neuropathic pain. This study examined the role of MARK1 or MARK2 in regulating neurosynaptic plasticity induced by neuropathic pain. A rat spinal nerve ligation (SNL) model was established to induce neuropathic pain. The role of MARKs in nociceptive regulation was assessed by genetically knocking down MARK1 or MARK2 in amygdala and systemic administration of PCC0105003, a novel small molecule MARK inhibitor. Cognitive function, anxiety-like behaviours and motor coordination capability were also examined in SNL rats. Synaptic remodelling-associated signalling changes were detected with electrophysiological recording, Golgi-Cox staining, western blotting and qRT-PCR. MARK1 and MARK2 expression levels in amygdala and spinal dorsal horn were elevated in SNL rats. MARK1 or MARK2 knockdown in amygdala and PCC0105003 treatment partially attenuated pain-like behaviours along with improving cognitive deficit, anxiogenic-like behaviours and motor coordination in SNL rats. Inhibition of MARKs signalling reversed synaptic plasticity at the functional and structural levels by suppressing NR2B/GluR1 and EB3/Drebrin signalling pathways both in amygdala and spinal dorsal horn. These results suggest that MARKs-mediated synaptic remodelling plays a key role in the pathogenesis of neuropathic pain and that pharmacological inhibitors of MARKs such as PCC0105003 could represent a novel therapeutic strategy for the management of neuropathic pain.

HSP27 Modulates Neuropathic Pain by Inhibiting P2X3 Degradation.

The role of heat shock protein 27 (HSP27), a chaperone, in neuropathic pain after nerve injury has not been systematically surveyed despite its neuroprotective and regeneration-promoting effects. In this study, we found that HSP27 expression in sensory neurons of the dorsal root ganglia (DRG) mediated nerve injury-induced neuropathic pain. Neuropathic pain behaviors were alleviated by silencing HSP27 in the DRG of a rat spinal nerve ligation (SNL) model. Local injection of an HSP27-overexpression construct into the DRG of naïve rats elicited neuropathic pain behaviors. HSP27 interacted with a purinergic receptor, P2X3, and their expression patterns corroborated the induction and reversal of neuropathic pain according to two lines of evidence: colocalization immunohistochemically and immunoprecipitation biochemically. In a cell model cotransfected with HSP27 and P2X3, the degradation rate of P2X3 was reduced in the presence of HSP27. Such an alteration was mediated by reducing P2X3 ubiquitination in SNL rats and was reversed after silencing HSP27 in the DRGs of SNL rats. In summary, the interaction of HSP27 with P2X3 provides a new mechanism of injury-induced neuropathic pain that could serve as an alternative therapeutic target.

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.

Three-dimensional analysis of the characteristics of joint motion and gait pattern in a rodent model following spinal nerve ligation

BackgroundThe spinal nerve ligation (SNL) rat is well known as the most common rodent model of neuropathic pain without motor deficit. Researchers have performed analyses using only the von Frey and thermal withdrawal tests to evaluate pain intensity in the rat experimental model. However, these test are completely different from the neurological examinations performed clinically. We think that several behavioral reactions must be observed following SNL because the patients with neuropathic pain usually have impaired coordination of the motions of the right–left limbs and right–left joint motion differences. In this study, we attempted to clarify the pain behavioral reactions in SNL rat model as in patients. We used the Kinema-Tracer system for 3D kinematics gait analysis to identify new characteristic parameters of each joint movement and gait pattern.ResultsThe effect of SNL on mechanical allodynia was a 47 ± 6.1% decrease in the withdrawal threshold during 1–8 weeks post-operation. Sagittal trajectories of the hip, knee and ankle markers in SNL rats showed a large sagittal fluctuation of each joint while walking. Top minus bottom height of the left hip and knee that represents instability during walking was significantly larger in the SNL than sham rats. Both-foot contact time, which is one of the gait characteristics, was significantly longer in the SNL versus sham rats: 1.9 ± 0.15 s vs. 1.03 ± 0.15 s at 4 weeks post-operation (p = 0.003). We also examined the circular phase time to evaluate coordination of the right and left hind-limbs. The ratio of the right/left circular time was 1.0 ± 0.08 in the sham rats and 0.62 ± 0.15 in the SNL rats at 4 weeks post-operation.ConclusionsWe revealed new quantitative parameters in an SNL rat model that are directly relevant to the neurological symptoms in patients with neuropathic pain, in whom the von Frey and thermal withdrawal tests are not used at all clinically. This new 3D analysis system can contribute to the analysis of pain intensity of SNL rats in detail similar to human patients’ reactions following neuropathic pain.

Protopanaxadiol alleviates neuropathic pain by spinal microglial dynorphin A expression following glucocorticoid receptor activation.

New remedies are required for the treatment of neuropathic pain due to insufficient efficacy of available therapies. This study provides a novel approach to develop painkillers for chronic pain treatment. The rat formalin pain test and spinal nerve ligation model of neuropathic pain were used to evaluate antinociception of protopanaxadiol. Primary cell cultures, immunofluorescence staining, and gene and protein expression were also performed for mechanism studies. Gavage protopanaxadiol remarkably produces pain antihypersensitive effects in neuropathic pain, bone cancer pain and inflammatory pain, with efficacy comparable with gabapentin. Long-term PPD administration does not induce antihypersensitive tolerance, but prevents and reverses the development and expression of morphine analgesic tolerance. Oral protopanaxadiol specifically stimulates spinal expression of dynorphin A in microglia but not in astrocytes or neurons. Protopanaxadiol gavage-related pain antihypersensitivity is abolished by the intrathecal pretreatment with the microglial metabolic inhibitor minocycline, dynorphin antiserum or specific κ-opioid receptor antagonist GNTI. Intrathecal pretreatment with glucocorticoid receptor)antagonists RU486 and dexamethasone-21-mesylate, but not GPR-30 antagonist G15 or mineralocorticoid receptor antagonist eplerenone, completely attenuates protopanaxadiol-induced spinal dynorphin A expression and pain antihypersensitivity in neuropathic pain. Treatment with protopanaxadiol, the glucocorticoid receptor agonist dexamethasone and membrane-impermeable glucocorticoid receptor agonist dexamethasone-BSA in cultured microglia induces remarkable dynorphin A expression, which is totally blocked by pretreatment with dexamthasone-21-mesylate. All the results, for the first time, indicate that protopanaxadiol produces pain antihypersensitivity in neuropathic pain probably through spinal microglial dynorphin A expression after glucocorticoid receptor activation and hypothesize that microglial membrane glucocorticoid receptor/dynorphin A pathway is a potential target to discover and develop novel painkillers in chronic pain.

Effect of Massage on the TLR4 Signalling Pathway in Rats with Neuropathic Pain.

This study set out to investigate the effect of massage on the Toll-like receptor 4 (TLR4) signalling pathway in the dorsal root ganglia of rats that had undergone spinal nerve ligation (SNL), with the hypothesis that massage could be used as an analgesic. Forty female SD rats were randomly divided into 5 groups: the control group, sham-operated group, model group, sham massage group, and massage group. There were 8 rats in each group. SNL rat models were established in the model group, sham massage group, and massage group. Rats in the sham-operated group underwent surgery to expose the vertebral nerves, but no further procedures were performed. The control group consisted of intact animals. The rats in the massage group underwent massage using a massage simulation machine once a day for 14 d in succession; the hind limbs of the rats in the sham massage group were gently touched with a cloth bag once a day for 14 continuous days. The rats in the control group, the sham-operated group, and the model group did not receive any intervention and were observed for 14 d. Paw withdrawal thermal latency (PWTL) and paw withdrawal mechanical threshold (PWMT) of rats in each group were detected 1 d before modelling and at 1, 3, 7, and 14 d after modelling. Fourteen days after modelling, the expression levels of TLR4, IRAK1, TRAF6, TNF-α, and IL-6 were detected in all rats. The PWTL and PWMT of SNL rats were decreased, while these parameters were elevated after massage. SNL rats showed higher levels of TLR4, IRAK1, TRAF6, IL-6, and TNF-α, and massage effectively lowered the expression levels of these molecules. Inhibiting activation of the TLR4 signalling pathway, which can reduce the release of inflammatory factors, may be one mechanism by which massage treats neuropathic pain.

Adipose Tissue-Derived Stem Cells Alleviate Cold Allodynia in a Rat Spinal Nerve Ligation Model of Neuropathic Pain.

Neuropathic pain caused by lesions or nervous system dysfunction is a neuroimmune disease with limited therapeutic options. Adipose tissue-derived stem cells (ASCs) are multipotent mesenchymal stem cells with potent immunosuppressive properties, and their use as novel cell-based therapeutics have been proposed in many immune diseases. However, the analgesic effect and efficacy of ASCs to treat neuropathic pain remain unclear. This study, thus, investigated whether ASCs or ASC-derived culture medium can relieve neuropathic pain behaviors (i.e., mechanical and cold allodynia) in a rat model with L5 spinal nerve ligation. Intrathecal injection of ASCs significantly reduced cold allodynia, but not mechanical allodynia. Importantly, cold allodynia was completely reversed in rats with repeated injections of ASCs. In contrast, intrathecal injection of ASC-derived culture medium or retro-orbital injection of ASCs had no effect on neuropathic pain behaviors. These results suggest a novel and alternative therapeutic application of ASCs to target specific neuropathic pain behaviors.

Loss of SNHG4 Attenuated Spinal Nerve Ligation-Triggered Neuropathic Pain through Sponging miR-423-5p.

Neuropathic pain is an intractable comorbidity of spinal cord injury. Increasing noncoding RNAs have been implicated in neuropathic pain development. lncRNAs have been recognized as significant regulators of neuropathic pain. lncRNA Small Nucleolar RNA Host Gene 4 (SNHG4) is associated with several tumors. However, the molecular mechanisms of SNHG4 in neuropathic pain remain barely documented. Here, we evaluated the function of SNHG4 in spinal nerve ligation (SNL) rat models. We observed that SNHG4 was significantly upregulated in SNL rat. Knockdown of SNHG4 was able to attenuate neuropathic pain progression via regulating behaviors of neuropathic pain including mechanical and thermal hyperalgesia. Moreover, knockdown of SNHG4 could repress the neuroinflammation via inhibiting IL-6, IL-12, and TNF-α while inducing IL-10 levels. Additionally, miR-423-5p was predicted as the target of SNHG4 by employing bioinformatics analysis. miR-423-5p has been reported to exert significantly poorer in several diseases. However, the role of miR-423-5p in the development of neuropathic pain is needed to be clarified. Here, in our investigation, RIP assay confirmed the correlation between miR-423-5p and SNHG4. Meanwhile, we found that miR-423-5p was significantly decreased in SNL rat models. SNHG4 regulated miR-423-5p expression negatively. As exhibited, the loss of miR-423-5p contributed to neuropathic pain progression, which was rescued by the silence of SNHG4. Therefore, our study indicated SNHG4 as a novel therapeutic target for neuropathic pain via sponging miR-423-5p.

Knockdown of Linc00052 alleviated spinal nerve ligation-triggered neuropathic pain through regulating miR-448 and JAK1.

The dysfunction of the nervous system contributes to neuropathic pain. Long noncoding RNAs are reported to participate in neuropathic pain. Recently, Linc00052 is implicated to be closely associated with multiple diseases. Nevertheless, the mechanisms of Linc00052 remain barely explored in neuropathic pain development. Currently, spinal nerve ligation (SNL) triggered neuropathic pain was employed in our investigation. Here, we assessed the function of Linc00052 in SNL rat models. Interestingly, we reported Linc00052 was significantly elevated in SNL rats. Loss of Linc00052 could reduce neuropathic pain progression via regulating the behaviors of neuropathic pain. Additionally, knockdown of Linc00052 repressed the processes of neuroinflammation. Interleukin (IL)-6 and tumor necrosis factor α level were inhibited while IL-10 was induced by the silence of Linc00052. Moreover, we predicted miR-448 can serve as a target of Linc00052. miR-448 exerts a crucial power in several diseases. Currently, we exhibited miR-448 was remarkably downregulated in SNL rats. RNA immunoprecipitation experiments validated the association between miR-448 and Linc00052. Inhibition of Linc00052 could reverse the roles of miR-448 on neuropathic pain development. Furthermore, Janus kinase 1 (JAK1) was displayed as the putative target of miR-448 in the present investigation. It was showed that JAK1 was induced in SNL rats. Loss of miR-448 could dramatically induce the expression of JAK1, which was rescued by knockdown of Linc00052. Taken these together, our study implied that Linc00052 functioned as a novel target of neuropathic pain via sponging miR-448 and regulating JAK1.

Evans Blue Reduces Neuropathic Pain Behavior by Inhibiting Spinal ATP Release.

Upon peripheral nerve injury, vesicular ATP is released from damaged primary afferent neurons. This extracellular ATP subsequently activates purinergic receptors of the spinal cord, which play a critical role in neuropathic pain. As an inhibitor of the vesicular nucleotide transporter (VNUT), Evans blue (EB) inhibits the vesicular storage and release of ATP in neurons. Thus, we tested whether EB could attenuate neuropathic pain behavior induced by spinal nerve ligation (SNL) in rats by targeting VNUT. An intrathecal injection of EB efficiently attenuated mechanical allodynia for five days in a dose-dependent manner and enhanced locomotive activity in an SNL rat model. Immunohistochemical analysis showed that EB was found in VNUT immunoreactivity on neurons in the dorsal root ganglion and the spinal dorsal horn. The level of ATP in cerebrospinal fluid in rats with SNL-induced neuropathic pain decreased upon administration of EB. Interestingly, EB blocked ATP release from neurons, but not glial cells in vitro. Eventually, the loss of ATP decreased microglial activity in the ipsilateral dorsal horn of the spinal cord, followed by a reduction in reactive oxygen species and proinflammatory mediators, such as interleukin (IL)-1β and IL-6. Finally, a similar analgesic effect of EB was demonstrated in rats with monoiodoacetate-induced osteoarthritis (OA) pain. Taken together, these data demonstrate that EB prevents ATP release in the spinal dorsal horn and reduces the ATP/purinergic receptor-induced activation of spinal microglia followed by a decline in algogenic substances, thereby relieving neuropathic pain in rats with SNL.

CXCL12/CXCR4 signaling contributes to neuropathic pain via central sensitization mechanisms in a rat spinal nerve ligation model.

SummaryBackgroundPrevious studies have demonstrated that the CXCL12/CXCR4 signaling axis is involved in the regulation of neuropathic pain (NP). Here, we performed experiments to test whether the CXCL12/CXCR4 signaling pathway contributes to the pathogenesis of neuropathic pain after spinal nerve ligation (SNL) via central sensitization mechanisms.MethodsNeuropathic pain was induced and assessed in a SNL rat model. The expression and distribution of CXCL12 or CXCR4 were examined by immunofluorescence staining and western blot. The effects of CXCL12 rat peptide, CXCL12 neutralizing antibody, CXCR4 antagonist, and astrocyte metabolic inhibitor on pain hypersensitivity were explored by behavioral tests in naive or SNL rats. We measured the expression level of c‐Fos and CGRP to evaluate the sensitization of neurons by RT‐PCR. The activation of astrocyte and microglia was analyzed by measuring the level of GFAP and iba‐1. The mRNA levels of the pro‐inflammatory cytokines such as TNF‐α, IL‐1β, and IL‐6 and Connexin 30, Connexin 43, EAAT 1, EAAT 2 were also detected by RT‐PCR.ResultsFirst, we found that the expression of CXCL12 and CXCR4 was upregulated after SNL. CXCL12 was mainly expressed in the neurons while CXCR4 was expressed both in astrocytes and neurons in the spinal dorsal horn after SNL. Moreover, intrathecal administration of rat peptide, CXCL12, induced hypersensitivity in naive rats, which was partly reversed by fluorocitrate. In addition, the CXCL12 rat peptide increased mRNA levels of c‐Fos, GFAP, and iba‐1. A single intrathecal injection of CXCL12 neutralizing antibody transiently reversed neuropathic pain in the SNL rat model. Consecutive use of CXCL12 neutralizing antibody led to significant delay in the induction of neuropathic pain, and reduced the expression of GFAP and iba‐1 in the spinal dorsal horn. Finally, repeated intrathecal administration of the CXCR4 antagonist, AMD3100, significantly suppressed the initiation and duration of neuropathic pain. The mRNA levels of c‐Fos, CGRP, GFAP, iba‐1, and pro‐inflammatory cytokines, also including Connexin 30 and Connexin 43 were decreased after injection of AMD3100, while EAAT 1 and EAAT 2 mRNAs were increased.ConclusionWe demonstrate that the CXCL12/CXCR4 signaling pathway contributes to the development and maintenance of neuropathic pain via central sensitization mechanisms. Importantly, intervening with CXCL12/CXCR4 presents an effective therapeutic approach to treat the neuropathic pain.

MicroRNA-7a ameliorates neuropathic pain in a rat model of spinal nerve ligation via the neurofilament light polypeptide-dependent signal transducer and activator of transcription signaling pathway.

Neuropathic pain is a type of chronic pain induced by either central or peripheral nerve injury. MicroRNAs have been recently linked to many diseases, including neuropathic pain. However, the role of miR-7a in neuropathic pain still remains elusive. Thus, we aim to investigate the effects of miR-7a on neuropathic pain based on the spinal nerve ligation rat model. After establishment of spinal nerve ligation rat models, rats were infected with adeno-associated virus-neurofilament light polypeptide, adeno-associated virus-miR-7a or treated with metformin. The paw withdrawal threshold and paw withdrawal latency were assessed afterward, and the expression of miR-7a and neurofilament light polypeptide as well as their interaction was determined. Subsequently, miR-7a was overexpressed or silenced in dorsal root ganglion cells to investigate the role of miR-7a in neuropathic pain. Furthermore, the regulatory effect of neurofilament light polypeptide on neuropathic pain was detected using plasmid overexpressing neurofilament light polypeptide. Spinal nerve ligation rat model exhibited upregulation of neurofilament light polypeptide but downregulation of miR-7a. In addition, neurofilament light polypeptide accumulation or miR-7a inhibition decreased paw withdrawal threshold and paw withdrawal latency. Then, neurofilament light polypeptide accumulation or miR-7a inhibition was observed to increase the phosphorylation level of signal transducer and activator of transcription. miR-7a was found to directly target neurofilament light polypeptide and downregulate neurofilament light polypeptide. In addition, inhibiting the signal transducer and activator of transcription signaling pathway was also revealed to increase paw withdrawal threshold and paw withdrawal latency. Collectively, our study demonstrated that miR-7a ameliorated neuropathic pain via blocking the signal transducer and activator of transcription signaling pathway by repressing neurofilament light polypeptide. These findings, if taken further, can be of important clinical significance in treating patients with neuropathic pain.

Synergistic interaction between the agonism of cebranopadol at nociceptin/orphanin FQ and classical opioid receptors in the rat spinal nerve ligation model.

Cebranopadol (trans‐6′‐fluoro‐4′,9′‐dihydro‐N,N‐dimethyl‐4‐phenyl‐spiro[cyclohexane‐1,1′(3′H)‐pyrano[3,4‐b]indol]‐4‐amine) is a novel analgesic nociceptin/orphanin FQ opioid peptide (NOP) and classical opioid receptor (MOP, DOP, and KOP) agonist with highly efficacious and potent activity in a broad range of rodent models of nociceptive, inflammatory, and neuropathic pain as well as limited opioid‐type side effects such as respiratory depression. This study was designed to explore contribution and interaction of NOP and classical opioid receptor agonist components to cebranopadol analgesia in the rat spinal nerve ligation (SNL) model. Assessing antihypersensitive activity in SNL rats intraperitoneal (IP) administration of cebranopadol resulted in ED 50 values of 3.3 and 3.58 μg/kg in two independent experiments. Pretreatment (IP) with J‐113397 (4.64 mg/kg) a selective antagonist for the NOP receptor or naloxone (1 mg/kg), naltrindole (10 mg/kg), or nor‐BNI (10 mg/kg), selective antagonists for MOP, DOP, and KOP receptors, yielded ED 50 values of 14.1, 16.9, 17.3, and 15 μg/kg, respectively. This 4‐5 fold rightward shift of the dose‐response curves suggested agonistic contribution of all four receptors to the analgesic activity of cebranopadol. Combined pretreatment with a mixture of the antagonists for the three classical opioid receptors resulted in an 18‐fold potency shift with an ED 50 of 65.5 μg/kg. The concept of dose equivalence was used to calculate the expected additive effects of the parent compound for NOP and opioid receptor contribution and to compare them with the observed effects, respectively. This analysis revealed a statistically significant difference between the expected additive and the observed effects suggesting intrinsic synergistic analgesic interaction of the NOP and the classical opioid receptor components of cebranopadol. Together with the observation of limited respiratory depression in rats and humans the synergistic interaction of NOP and classical opioid receptor components in analgesia described in the current study may contribute to the favorable therapeutic index of cebranopadol observed in clinical trials.

Pro-resolving mediator maresin 1 ameliorates pain hypersensitivity in a rat spinal nerve ligation model of neuropathic pain.

BackgroundPro-resolving mediators (PRMs) are considered as emerging analgesics for chronic pain. Maresin 1 (MaR1) is a newly identified member of PRMs, and recent studies implicate its potential role in some pain conditions. As the function of MaR1 in neuropathic pain remains unclear, we investigated the effects of MaR1 on pain hypersensitivity and the underlying mechanism using a rat spinal nerve ligation (SNL) model of neuropathic pain.Materials and methodsMaR1 (100 ng/10 μL) or commensurable artificial cerebrospinal fluid was delivered via intrathecal catheter from days 3 to 5 post-SNL followed by assessment of mechanical allodynia and thermal hyperalgesia. Ipsilateral L4–L5 spinal cord tissue was collected on day 7 post-SNL and assessed by Western blotting, enzyme-linked immunosorbent assay or immunohistochemistry.ResultsIntrathecal MaR1 significantly attenuated mechanical allodynia and thermal hyperalgesia from day 5 to day 7 post-SNL, which was associated with decreased spinal levels of glial markers, GFAP and IBA1. It was also found that intrathecal MaR1 downregulated phosphorylation levels of NF-κB p65 and its nuclear translocation, as well as decreased protein levels of pro-inflammatory cytokines, TNF-α, IL-1β and IL-6. Further, MaR1 treatment restored PSD95 and synapsin II levels, suggesting that MarR1 also protected synaptic integrity.ConclusionOur results indicate that MaR1 ameliorates the SNL-induced neuropathic pain by regulating glial activities and pro-inflammatory cytokines release. The present study offers insight into the potential of MaR1 as a novel intervention to ameliorate neuropathic pain.

Selectivity profiling of NOP, MOP, DOP and KOP receptor antagonists in the rat spinal nerve ligation model of mononeuropathic pain

Agonists selectively acting at NOP, MOP, DOP and KOP receptors as well as mixed opioid receptor agonists are known to exert anti-hypersensitive efficacy in the rat spinal nerve ligation (SNL) model of neuropathic pain. To investigate the relative contribution of individual opioid receptor activation to the overall efficacy of mixed opioid receptor agonists, selective doses of respective opioid receptor antagonists have to be employed. In order to identify such selective antagonist doses, doses of the selective NOP, MOP, DOP and KOP receptor agonists Ro65–6570, morphine, SNC-80 and U50488H, that produced maximum efficacy without apparent side effects, were challenged by each of the receptor antagonists J-113397 (NOP receptor), naloxone (MOP receptor), naltrindole (DOP receptor) and nor-binaltorphimine (KOP receptor). J-113397, naloxone, naltrindole and nor-binaltorphimine at intraperitoneal doses of 4.64, 1, 10, and 10 mg/kg, respectively, inhibited anti-hypersensitive effects mediated by the corresponding cognate NOP, MOP, DOP and KOP receptor selective agonists. Selectivity could be demonstrated for MOP, DOP and NOP receptor antagonists, as they did not attenuate effects mediated by agonists acting on non-cognate receptors, whereas the KOP receptor antagonist nor-BNI demonstrated partial cross-antagonism of the DOP receptor agonist SNC-80. Thus, specific doses of opioid receptor antagonists that completely but still selectively attenuate full anti-hypersensitive efficacy of corresponding opioid receptor agonists were identified in the rat SNL model.

Neuropathic pain-alleviating effects of pyrazole-conjugated arylsulfonamides as 5-HT6 receptor antagonists

A novel series of arylsulfonylaminomethyl-3-(1-phenyl-5-isopropyl)pyrazoles was evaluated for serotonin receptor subtype 6 (5-HT6R) antagonistic effects in vitro. We also investigated their neuropathic pain-alleviating effects in vivo using a rat spinal nerve ligation (SNL) model. Bicyclic aromatic sulfonamino groups, such as naphthalene and quinolin-substituted derivatives, showed good 5-HT6 inhibitory activity in vitro. Among them, selected compounds, 12 and 13, having 8-quinoylsulfonamino groups, showed potent neuropathic pain-alleviating effects in the rat model.

5-HT2C Receptor Knockdown in the Amygdala Inhibits Neuropathic-Pain-Related Plasticity and Behaviors.

Neuroplasticity in the amygdala drives pain-related behaviors. The central nucleus (CeA) serves major amygdala output functions and can generate emotional-affective behaviors and modulate nocifensive responses. The CeA receives excitatory and inhibitory inputs from the basolateral nucleus (BLA) and serotonin receptor subtype 5-HT2CR in the BLA, but not CeA, has been implicated anxiogenic behaviors and anxiety disorders. Here, we tested the hypothesis that 5-HT2CR in the BLA plays a critical role in CeA plasticity and neuropathic pain behaviors in the rat spinal nerve ligation (SNL) model. Local 5-HT2CR knockdown in the BLA with stereotaxic injection of 5-HT2CR shRNA AAV vector decreased vocalizations and anxiety- and depression-like behaviors and increased sensory thresholds of SNL rats, but had no effect in sham controls. Extracellular single-unit recordings of CeA neurons in anesthetized rats showed that 5-HT2CR knockdown blocked the increase in neuronal activity (increased responsiveness, irregular spike firing, and increased burst activity) in SNL rats. At the synaptic level, 5-HT2CR knockdown blocked the increase in excitatory transmission from BLA to CeA recorded in brain slices from SNL rats using whole-cell patch-clamp conditions. Inhibitory transmission was decreased by 5-HT2CR knockdown in control and SNL conditions to a similar degree. The findings can be explained by immunohistochemical data showing increased expression of 5-HT2CR in non-GABAergic BLA cells in SNL rats. The results suggest that increased 5-HT2CR in the BLA contributes to neuropathic-pain-related amygdala plasticity by driving synaptic excitation of CeA neurons. As a rescue strategy, 5-HT2CR knockdown in the BLA inhibits neuropathic-pain-related behaviors.SIGNIFICANCE STATEMENT Neuroplasticity in the amygdala has emerged as an important pain mechanism. This study identifies a novel target and rescue strategy to control abnormally enhanced amygdala activity in an animal model of neuropathic pain. Specifically, an integrative approach of gene transfer, systems and brain slice electrophysiology, behavior, and immunohistochemistry was used to advance the novel concept that serotonin receptor subtype 5-HT2C contributes critically to the imbalance between excitatory and inhibitory drive of amygdala output neurons. Local viral vector-mediated 5-HT2CR knockdown in the amygdala normalizes the imbalance, decreases neuronal activity, and inhibits neuropathic-pain-related behaviors. The study provides valuable insight into serotonin receptor (dys)function in a limbic brain area.

Anti-allodynic effect of intrathecal processed Aconitum jaluense is associated with the inhibition of microglial activation and P2X7 receptor expression in spinal cord

BackgroundFor their analgesic and anti-arthritic effects, Aconitum species have been used in folk medicine in some East Asian countries. Although their analgesic effect is attributed to its action on voltage-dependent sodium channels, they also suppress purinergic receptor expression in dorsal root ganglion neurons in rats with neuropathic pain. In vitro study also demonstrated that the Aconitum suppresses ATP-induced P2X7 receptor (P2X7R)-mediated inflammatory responses in microglial cell lines. Herein, we examined the effect of intrathecal administration of thermally processed Aconitum jaluense (PA) on pain behavior, P2X7R expression and microglial activation in a rat spinal nerve ligation (SNL) model.MethodsMechanical allodynia induced by L5 SNL in Sprague-Dawley rats was measured using the von Frey test to evaluate the effect of intrathecal injection of PA. Changes in the expression of P2X7R in the spinal cord were examined using RT-PCR and Western blot analysis. In addition, the effect of intrathecal PA on microglial activation was evaluated by immunofluorescence.ResultsIntrathecal PA attenuated mechanical allodynia in a dose-dependent manner showing both acute and chronic effects with 65 % of the maximal possible effect. The expression and production of spinal P2X7R was increased five days after SNL, but daily intrathecal PA injection significantly inhibited the increase to the level of naïve animals. Immunofluorescence of the spinal cord revealed a significant increase in P2X7R expression and activation of microglia in the dorsal horn, which was inhibited by intrathecal PA treatment. P2X7R co-localized with microglia marker, but not neurons.ConclusionsIntrathecal PA exerts anti-allodynic effects in neuropathic pain, possibly by suppressing P2X7R production and expression as well as reducing microglial activation in the spinal cord.