Rat Model Of Neuropathic Pain Research Articles

Sex Differences in CGRP Regulation and Function in the Amygdala in a Rat Model of Neuropathic Pain

The amygdala has emerged as a key player in the emotional response to pain and pain modulation. The lateral and capsular regions of the central nucleus of the amygdala (CeA) represent the “nociceptive amygdala” due to their high content of neurons that process pain-related information. These CeA divisions are the targets of the spino-parabrachio-amygdaloid pain pathway, which is the predominant source of calcitonin gene-related peptide (CGRP) within the amygdala. Changes in lateral and capsular CeA neurons have previously been observed in pain models, and synaptic plasticity in these areas has been linked to pain-related behavior. CGRP has been demonstrated to play an important role in peripheral and spinal mechanisms, and in pain-related amygdala plasticity in male rats in an acute arthritis pain model. However, the role of CGRP in chronic neuropathic pain-related amygdala function and behaviors remains to be determined for both male and female rats. Here we tested the hypothesis that the CGRP1 receptor is involved in neuropathic pain-related amygdala activity, and that blockade of this receptor can inhibit neuropathic pain behaviors in both sexes. CGRP mRNA expression levels in the CeA of male rats were upregulated at the acute stage of the spinal nerve ligation (SNL) model of neuropathic pain, whereas female rats had significantly higher CGRP and CGRP receptor component expression at the chronic stage. A CGRP1 receptor antagonist (CGRP 8-37) administered into the CeA in chronic neuropathic rats reduced mechanical hypersensitivity (von Frey and paw compression tests) in both sexes but showed female-predominant effects on emotional-affective responses (ultrasonic vocalizations) and anxiety-like behaviors (open field test). CGRP 8-37 inhibited the activity of CeA output neurons assessed with calcium imaging in brain slices from chronic neuropathic pain rats. Together, these findings may suggest that CGRP1 receptors in the CeA are involved in neuropathic pain-related amygdala activity and contribute to sensory aspects in both sexes but to emotional-affective pain responses predominantly in females. The sexually dimorphic function of CGRP in the amygdala would make CGRP1 receptors a potential therapeutic target for neuropathic pain relief, particularly in females in chronic pain conditions.

Response After Repeated Ketamine Injections in a Rat Model of Neuropathic Pain.

Ketamine, an N-methyl-D-aspartate antagonist, reduces pain by decreasing central sensitization and pain windup. However, chronic ketamine use can cause tolerance, dependency, impaired consciousness, urinary symptoms, and abdominal pain. This study aimed to investigate the effects of repeated ketamine injections and ketamine readministration after discontinuation in a rat model of neuropathic pain. To induce neuropathic pain, partial sciatic nerve ligation (PSNL) was performed in 15 male Wistar rats, and these animals were divided into three groups: PSNL (control), PSNL + ketamine 5 mg/kg (K5), and PSNL + ketamine 10 mg/kg (K10; n=5 each). Ketamine was injected intraperitoneally daily for 4 weeks, discontinued for 2 weeks, and then readministered for 1 week. Following PSNL, the mechanical withdrawal threshold was determined weekly using the Von Frey. The K10 group showed a significant increase in the mechanical withdrawal threshold, presented here as the target force (in g), at 21 and 28 days compared to the time point before ketamine injection (mean±SE, 276.0±24.0 vs. 21.6±2.7 and 300.0±0.0 vs. 21.6±2.7, respectively; P<0.01) and at 14, 21, and 28 days compared to the control group (108.2±51.2 vs. 2.7±1.3, 276.0±24.0 vs. 2.5±1.5, and 300.0±0.0 vs. 4.0±0.0, respectively; P<0.05). However, in the K10 group, the ketamine effects decreased significantly at 7 days after readministration compared to those after 28 days of repeated injections (P<0.05). In the K10 group, repeated ketamine injections showed a significant increase in antinociceptive effect for >2 weeks, but this ketamine effect decreased after drug readministration.

Protein Tyrosine Phosphatase Receptor Type D Regulates Neuropathic Pain After Nerve Injury via the STING-IFN-I Pathway.

Neuropathic pain is usually caused by injury or dysfunction of the somatosensory system, and medicine is a common way of treatment. Currently, there are still no satisfactory drugs, like opioids and lidocaine, which carry a high risk of addiction. Protein tyrosine phosphatase receptor type D (PTPRD) is a known therapeutic target in addiction pathways and small molecule inhibitors targeting it, such as 7-butoxy illudalic acid analog (7-BIA), have recently been developed to tackle addition. PTPRD is also upregulated in the dorsal root ganglion (DRG) in a rat model of neuropathic pain, but is not yet clear whether PTPRD contributes to the development of neuropathic pain. Here, we established a chronic constriction injury (CCI) and evaluated PTPRD expression and its association with neuropathic pain. PTPRD expression was found to gradually increase after CCI in DRGs, and its expression was concomitant with the progressive development of hypersensitivity as assessed by both mechanical and thermal stimuli. Both PTPRD knockdown and administration of PTPRD inhibitor 7-BIA alleviated CCI-induced neuropathic pain while upregulating STING and IFN-α in the DRG. Treatment with H-151, a STING inhibitor, abolished the analgesic effects of PTPRD knockdown. Taken together, our study suggests that increased levels of PTPRD in the DRG following CCI are involved in the development of neuropathic pain via the STING-IFN-I pathway. 7-BIA, a small molecule inhibitor of PTPRD with anti-addiction effects, may represent a novel and safe therapeutic strategy for the clinical management of neuropathic pain without the risk of addiction.

Involvement of Opioid Peptides in the Analgesic Effect of Spinal Cord Stimulation in a Rat Model of Neuropathic Pain.

Spinal cord stimulation (SCS)-induced analgesia was characterized, and its underlying mechanisms were examined in a spared nerve injury model of neuropathic pain in rats. The analgesic effect of SCS with moderate mechanical hypersensitivity was increased with increasing stimulation intensity between the 20% and 80% motor thresholds. Various frequencies (2, 15, 50, 100, 10000 Hz, and 2/100Hz dense-dispersed) of SCS were similarly effective. SCS-induced analgesia was maintained without tolerance within 24h of continuous stimulation. SCS at 2Hz significantly increased methionine enkephalin content in the cerebrospinal fluid. The analgesic effect of 2Hz was abolished by μ or κ opioid receptor antagonist. The effect of 100Hz was prevented by a κ antagonist, and that of 10kHz was blocked by any of the μ, δ, or κ receptor antagonists, suggesting that the analgesic effect of SCS at different frequencies is mediated by different endorphins and opioid receptors.

Sirtuin 2 Alleviates Chronic Neuropathic Pain by Suppressing Ferroptosis in Rats.

Neuropathic pain (NP) is chronic and associated with poor effects of general analgesia. It affects patients’ health and quality of life. The apoptotic process of lipid peroxidation caused by iron overload is called ferroptosis, which may be associated with nervous system disease. A recent study has found that sirtuin 2 (SIRT2) achieves a neuroprotective effect by suppressing ferroptosis. Herein, we aimed to examine whether SIRT2 regulated spared nerve injury (SNI)-induced NP by suppressing ferroptosis in rats. A rat model of NP was induced in adult male Sprague-Dawley rats weighing 200–250 g. Mechanical allodynia was observed from the first day after SNI and continued for 14 days. Compared with age-matched control rats, the expression of SIRT2 and ferroportin 1 (FPN1) decreased in the L4-6 spinal cord of the SNI-induced NP rats. In addition, we observed that the levels of both iron and anti-acyl-coenzyme A synthetase long-chain family member 4 (ACSL4) were significantly increased in the spinal cord after SNI, while the expression of glutathione peroxidase 4 (GPX4) was decreased. Furthermore, an intrathecal injection of SIRT2 overexpressed recombinant adenovirus, which upregulated the expression of SIRT2, attenuated mechanical allodynia, enhanced the level of FPN1, inhibited intracellular iron accumulation, and reduced oxidant stress levels, thereby reversing the changes to ACSL4 and GPX4 expression in the SNI rats. This evidence suggests that SIRT2-targeted therapeutics may help relieve the symptoms of chronic NP.

Evaluation of [18F]F-DPA PET for Detecting Microglial Activation in the Spinal Cord of a Rat Model of Neuropathic Pain

PurposeRecent studies have linked activated spinal glia to neuropathic pain. Here, using a positron emission tomography (PET) scanner with high spatial resolution and sensitivity, we evaluated the feasibility and sensitivity of N,N-diethyl-2-(2-(4-([18F]fluoro)phenyl)-5,7-dimethylpyrazolo[1,5-a] pyrimidin-3-yl)acetamide ([18F]F-DPA) imaging for detecting spinal cord microglial activation after partial sciatic nerve ligation (PSNL) in rats.ProceduresNeuropathic pain was induced in rats (n = 20) by PSNL, and pain sensation tests were conducted before surgery and 3 and 7 days post-injury. On day 7, in vivo PET imaging and ex vivo autoradiography were performed using [18F]F-DPA or [11C]PK11195. Ex vivo biodistribution and PET imaging of the removed spinal cord were carried out with [18F]F-DPA. Sham-operated and PK11195-pretreated animals were also examined.ResultsMechanical allodynia was confirmed in the PSNL rats from day 3 through day 7. Ex vivo autoradiography showed a higher lesion-to-background uptake with [18F]F-DPA compared with [11C]PK11195. Ex vivo PET imaging of the removed spinal cord showed [18F]F-DPA accumulation in the inflammation site, which was immunohistochemically confirmed to coincide with microglia activation. Pretreatment with PK11195 eliminated the uptake. The SUV values of in vivo [18F]F-DPA and [11C]PK11195 PET were not significantly increased in the lesion compared with the reference region, and were fivefold higher than the values obtained from the ex vivo data. Ex vivo biodistribution revealed a twofold higher [18F]F-DPA uptake in the vertebral body compared to that seen in the bone from the skull.Conclusions[18F]F-DPA aided visualization of the spinal cord inflammation site in PSNL rats on ex vivo autoradiography and was superior to [11C]PK11195. In vivo [18F]F-DPA PET did not allow for visualization of tracer accumulation even using a high-spatial-resolution PET scanner. The main reason for this result was due to insufficient SUVs in the spinal cord region as compared with the background noise, in addition to a spillover from the vertebral body.

Optogenetic manipulations of CeA-CRF neurons modulate pain- and anxiety-like behaviors in neuropathic pain and control rats

The amygdala plays a critical role in the emotional-affective component of pain and pain modulation. The central nucleus of amygdala (CeA) serves major output functions and has been linked to pain-related behaviors. Corticotropin releasing factor (CRF) in the CeA has emerged as an important modulator of pain and affective disorders. Here we measured the effects of optogenetic manipulation of CeA-CRF neurons on pain-related behaviors in a rat neuropathic pain model and under control conditions. Emotional-affective behaviors (vocalizations), mechanosensitivity (electronic von Frey anesthesiometer and calibrated forceps), and anxiety-like behaviors (open field test and elevated plus maze) were assessed in adult rats 1 week and 4 weeks after spinal nerve ligation (SNL model) and sham surgery (control). For optogenetic silencing or activation of CRF neurons, a Cre-inducible viral vector encoding enhanced halorhodopsin (eNpHR3.0) or channelrhodopsin 2 (ChR2) was injected stereotaxically into the right CeA of transgenic Crh-Cre rats. Light of the appropriate wavelength (590 nm for eNpHR3.0; 473 nm for ChR2) was delivered into the CeA with an LED optic fiber. Optical silencing of CeA-CRF neurons decreased the emotional-affective responses in the acute and chronic phases of the neuropathic pain model but had anxiolytic effects only at the chronic stage and no effect on mechanosensitivity. Optogenetic activation of CeA-CRF neurons increased the emotional-affective responses and induced anxiety-like behaviors but had no effect on mechanosensitivity in control rats. The data show the critical contribution of CeA-CRF neurons to pain-related behaviors under normal conditions and beneficial effects of inhibiting CeA-CRF neurons in neuropathic pain.This article is part of the Special Issue on ‘Advances in mechanisms and therapeutic targets relevant to pain’.

Discovery and Optimization of Biaryl Alkyl Ethers as a Novel Class of Highly Selective, CNS-Penetrable, and Orally Active Adaptor Protein-2-Associated Kinase 1 (AAK1) Inhibitors for the Potential Treatment of Neuropathic Pain.

Recent mouse knockout studies identified adapter protein-2-associated kinase 1 (AAK1) as a viable target for treating neuropathic pain. BMS-986176/LX-9211 (4), as a highly selective, CNS-penetrable, and potent AAK1 inhibitor, has advanced into phase II human trials. On exploring the structure-activity relationship (SAR) around this biaryl alkyl ether chemotype, several additional compounds were found to be highly selective and potent AAK1 inhibitors with good druglike properties. Among these, compounds 43 and 58 showed very good efficacy in two neuropathic pain rat models and had excellent CNS penetration and spinal cord target engagement. Both compounds also exhibited favorable physicochemical and oral pharmacokinetic (PK) properties. Compound 58, a central pyridine isomer of BMS-986176/LX-9211 (4), was 4-fold more potent than 4 in vitro and showed lower plasma exposure needed to achieve similar efficacy compared to 4 in the CCI rat model. However, both 43 and 58 showed an inferior preclinical toxicity profile compared to 4.

Analgesia with 5' extracellular nucleotidase-mediated electroacupuncture for neuropathic pain.

Acupuncture is a treatment for neuropathic pain, but its mechanism remains unclear. Previous studies showed that analgesia was induced in rats with neuropathic pain when their spinal cord adenosine content increased after electroacupuncture (EA); however, the mechanism behind this electroacupuncture-induced increase has not been clarified. This study aimed to determine the role that ecto-5'-nucleotidase plays in EA-induced analgesia for neuropathic pain. We performed electroacupuncture at the Zusanli acupoint on the seventh day after establishing a rat model of neuropathic pain induced through chronic constriction injuries. We observed the mechanical withdrawal threshold and thermal pain threshold and detected the expression of ecto-5'-nucleotidase in the spinal cord using Western blot. Chronic constriction injury rat models were intraperitoneally injected with α,β-methyleneadenosine 5'-diphosphate, an ecto-5'-nucleotidase inhibitor, 30 min before electroacupuncture. The adenosine content of the spinal cord was detected using high-performance liquid chromatography. Lastly, the adenosine A1 receptor agonist N6-cyclopentyladenosine was intrathecally injected into the lumbar swelling of the rats, and the mechanical withdrawal and thermal pain thresholds were reevaluated. Analgesia and increased ecto-5'-nucleotidase expression and adenosine content in the spinal cord were observed 1 h after electroacupuncture. α,β-methyleneadenosine 5'-diphosphate was able to inhibit upregulation of adenosine content and electroacupuncture-induced analgesia. After administration of N6-cyclopentyladenosine, electroacupuncture-induced analgesia was restored. Our results suggest that electroacupuncture at Zusanli can produce analgesia in chronic constriction injury rat models, possibly via the increased ecto-5'-nucleotidase expression induced through electroacupuncture, thus leading to increased adenosine expression in the spinal cord.

β-Lactam TRPM8 Antagonist RGM8-51 Displays Antinociceptive Activity in Different Animal Models.

Transient receptor potential melastatin subtype 8 (TRPM8) is a cation channel extensively expressed in sensory neurons and implicated in different painful states. However, the effectiveness of TRPM8 modulators for pain relief is still a matter of discussion, since structurally diverse modulators lead to different results, depending on the animal pain model. In this work, we described the antinociceptive activity of a β–lactam derivative, RGM8-51, showing good TRPM8 antagonist activity, and selectivity against related thermoTRP channels and other pain-mediating receptors. In primary cultures of rat dorsal root ganglion (DRG) neurons, RGM8-51 potently reduced menthol-evoked neuronal firing without affecting the major ion conductances responsible for action potential generation. This compound has in vivo antinociceptive activity in response to cold, in a mouse model of oxaliplatin-induced peripheral neuropathy. In addition, it reduces cold, mechanical and heat hypersensitivity in a rat model of neuropathic pain arising after chronic constriction of the sciatic nerve. Furthermore, RGM8-51 exhibits mechanical hypersensitivity-relieving activity, in a mouse model of NTG-induced hyperesthesia. Taken together, these preclinical results substantiate that this TRPM8 antagonist is a promising pharmacological tool to study TRPM8-related diseases.

Blocking NLRP3 inflammasome expression by RAS-like protein A mitigates neuropathic pain in chronic constriction injury rat models

Purpose: To investigate the role of RAS-like protein A (RalA) in lipopolysaccharide-induced inflammatory regulation in primary microglia of chronic constriction injury (CCI)-induced neuropathic pain in rat models.&#x0D; Methods: In vitro, overexpression (OE) of RalA was performed in rat microglia using transfection procedure, and then LPS was used to provoke the inflammatory phenotype. In vivo, the rat model of neuropathic pain was induced using CCI and treated with LV-RalA. Neuroinflammatory levels including the expressions of IL-1β, IL-6, and TNF-α were detected. Moreover, the expressions of NF-κB p65, thioredoxin-interacting protein (TXNIP) and NLR family pyrin domain-containing 3(NLRP3)were examined in CCI rats and microglial cells. Finally, the functional evaluation was determined via mechanical allodynia and thermal hyperalgesia assays.&#x0D; Results: The level of RalA decreased in the dorsal horn following CCI. OE of RalA in microglia after LPS insult and CCI-induced rat model significantly decreased the expressions of inflammation promoters (p &lt; 0.05). Mechanistically, OE of RalA mitigated inflammatory response by inhibiting NF-κB/TXNIP/NLRP3 signaling pathway, thus attenuating neuropathic pain in microglial cells and CCI rats.&#x0D; Conclusion: These results indicate that the OE of RalA plays a protective role in CCI-induced neuropathic pain via NF-κB/TXNIP/ NLRP3 axis. These findings may provide a promising therapeutic target for neuropathic pain.

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.

Antinociceptive effect of N-acetyl glucosamine in a rat model of neuropathic pain.

This study was aimed at evaluating the efficacy of glucosamine and potential mechanisms of actions in a neuropathic pain model in rats. Glucosamine (500, 1000 and 2000 mg/kg) was administered via gavage route, 1 day before the chronic constriction injury (CCI) of sciatic nerve and daily for 14 days (prophylactic regimen), or from days 5 to 14 post-injury (therapeutic regimen), as the indicators of neuropathic pain, mechanical allodynia, cold allodynia and thermal hyperalgesia were assessed on days 0, 3, 5, 7, 10 and 14 after ligation. Inducible nitric oxide synthase (iNOS) and tumour necrosis factor alpha (TNF-α) gene expressions were measured by real-time polymerase chain reaction. TNF-α protein content was measured using the enzyme-linked immunosorbent assay method. Three days after nerve injury, the threshold of pain was declined among animals subjected to neuropathic pain. Mechanical and cold allodynia, as well as thermal hyperalgesia were attenuated by glucosamine (500, 1000, 2000 mg/kg) in the prophylactic regimen. However, existing pain was not decreased by this drug. Increased mRNA expression of iNOS and TNF-α was significantly reduced in the spinal cord of CCI animals by glucosamine (500, 1000, 2000 mg/kg) in the prophylactic regimen. The overall expression of spinal TNF-α was increased by CCI, but this increase was reduced in animals receiving glucosamine prophylactic treatment. Findings suggest that glucosamine as a safe supplement may be a useful candidate in preventing neuropathic pain following nerve injury. Antioxidant and anti-inflammatory effects may be at least in part responsible for the antinociceptive effects of this drug.

Involvement of the BDNF-TrkB-KCC2 pathway in neuropathic pain after brachial plexus avulsion.

IntroductionBrachial plexus avulsion significantly increased brain‐derived neurotrophic factor (BDNF) release in the spinal cord. Here we investigated the involvement of the BDNF–TrkB–KCC2 pathway in neuropathic pain caused by BPA injury. We hypothesized that activation of BDNF–TrkB may inhibit neuronal excitability by downregulating KCC2 to maintain a high intracellular Cl‐concentration. We established a neuropathic pain rat model by avulsion of the lower trunk brachial plexus, and investigated the effects of the TrkB‐specific antibody K‐252a on the expression of BDNF, TrkB, and KCC2.MethodsWe randomly divided 40 male SD rats into four groups. In the brachial plexus avulsion group, C8‐T1 roots were avulsed from the spinal cord at the lower trunk level. In the K252a group, 5uL K252a was applied intrathecally daily for three days after avulsion. In the sham surgery group, expose only and without damage. The control group did not undergo any treatment. Mechanical hyperalgesia and cold allodynia were analyzed by electronic pain measuring instrument and acetone spray method at different time points on days 1, 3, 7, 10, 14, and 21 after surgery. At 21 days after surgery, the expression of BDNF and TrkB in dorsal horn neurons and GFAP in astrocytes were detected by immunohistochemistry at the C5‐T1 segment of the spinal cord. The expression levels of BDNF, TrkB, and KCC2 in the C5‐T1 spinal cord were measured by Western Blot at 7 and 21 days.ResultsMechanical hyperalgesia and cold allodynia were significantly reduced in the K252a group compared with the brachial plexus avulsion group. Compared with the BPA group, BDNF, TrkB and GFAP were significantly decreased in the K252a group at 21 days after treatment by immunohistochemical test. In the WB test, the expressions of BDNF and TrkB in the K252a group were quantitatively detected to be decreased, while the expression of KCC2 was increased, which was obvious at 7 and 21 days.ConclusionBDNF‐TrkB‐KCC2 pathway can significantly relieve neuropathic pain after BPA, and is a potential target for the treatment of neuropathic pain.

Microglia inflammatory response contributes to chronic constriction injury-induced neuropathic pain via miR-339/PFKFB3 axis

&#x0D; &#x0D; &#x0D; &#x0D; Purpose: To investigate the effect of miR-339 on neuropathic pain.&#x0D; Methods: A rat neuropathic pain model was established through chronic constriction injury (CCI). Expression of miR-339 in spinal cord was determined 14 days later. Microglial inflammatory response was evaluated using immunofluorescence analysis of ionized calcium binding adaptor molecule 1 (Iba1), while IL-6 and TNF-α were assessed by enzyme-linked immunosorbent assay (ELISA). Pain- associated behavioral effects and microglia-related inflammation were investigated after intrathecal administration of miR-339 agomir into rats post-CCI. The target gene of miR-339 involved in neuropathic pain was evaluated by a luciferase reporter assay. Microglia cells were isolated from rats, then treated with lipopolysaccharide (LPS). The LPS-induced inflammatory response in microglia cells was determined using quantitative reverse transcription PCR analysis of IL-6 and TNF-α.&#x0D; Results: CCI decreased mechanical allodynia and thermal hyperalgesia thresholds, but increased Iba1, IL-6, and TNF-α in rats. MiR-339 was reduced in rat spinal cord after CCI induction while intrathecal injection of miR-339 agomir alleviated CCI-induced changes in mechanical and thermal hyperalgesia in rats, and reversed expression of Iba1, IL-6, and TNF-α. Furthermore, 6-phosphofructo-2- kinase/fructose-2,6-bisphosphatase-3 (PFKFB3) was identified as a miR-339 target gene, and over- expression of miR-339 decreased the expression of PFKFB3, IL-6, and TNF-α in LPS-induced microglia cells.&#x0D; Conclusion: The miR-339/PFKFB3 axis ameliorates CCI-induced neuropathic pain by suppression of microglia inflammatory response, suggesting a novel strategy for neuropathic pain management.&#x0D; &#x0D; &#x0D; &#x0D;

Improvement of the affinity of an anti-rat P2X4 receptor antibody by introducing electrostatic interactions

We have recently developed a mouse monoclonal antibody (12–10H) binding to the head domain region in rat P2X4 receptor (rP2X4R, which is crucial for the pathogenesis of neuropathic pain) expressed on the cell with the highest binding affinity (KD = 20 nM). However, the 12–10H antibody failed to detect endogenously expressed P2X4Rs in microglia isolated from the spinal cord of rats whose spinal nerves were injured. Then, we prepared R5 mutant, in which five arginine residues were introduced into variable regions except for the “hot spot” in the 12–10H antibody to increase electrostatic interactions with the head domain, an anionic region, in rP2X4R. The mutation resulted in an increase of 50-fold in the affinity of the R5 mutant for the head domain with respect to the intact 12–10H antibody. As a result, detection of P2X4Rs endogenously expressed on primary cultured microglial cells originated from the neonatal rat brain and spinal cord microglia isolated from a rat model of neuropathic pain was achieved. These findings suggest a strategy to improve the affinity of a monoclonal antibody for an anionic antigen by the introduction of several arginine residues into variable regions other than the “hot spot” in the paratope.

Pharmacological interactions between intrathecal pregabalin plus tianeptine or clopidogrel in a rat model of neuropathic pain.

BackgroundThere is still unmet need in treating neuropathic pain and increasing awareness regarding the use of drug combinations to increase the effectiveness of treatment and reduce adverse effects in patients with neuropathic pain.MethodsThis study was performed to determine the individual and combined effects of pregabalin, tianeptine, and clopidogrel in a rat model of neuropathic pain. The model was created by ligation of the L5-L6 spinal nerve in male Sprague–Dawley rats; mechanical allodynia was confirmed using von Frey filaments. Drugs were administered to the intrathecal space and mechanical allodynia was assessed; drug interactions were estimated by isobolographic or fixed-dose analyses.ResultsIntrathecal pregabalin and tianeptine increased the mechanical withdrawal threshold in a dose-dependent manner, but intrathecal clopidogrel had little effect on the mechanical withdrawal threshold. An additive effect was noted between pregabalin and tianeptine, but not between pregabalin and clopidogrel.ConclusionsThese findings suggest that intrathecal coadministration of pregabalin and tianeptine effectively attenuated mechanical allodynia in the rat model of neuropathic pain. Thus, pregabalin plus tianeptine may be a valid option to enhance the efficacy of neuropathic pain treatment.

Exosomes carried miR-181c-5p alleviates neuropathic pain in CCI rat models.

Mesenchymal stem cells (MSCs) derived exosomes (Exos) are one of the most promising candidate for the treatment of this condition. However, the underlying molecular mechanism remains uncertain. Here we investigated the therapeutic effect of exosomal miR-181c-5p (ExomiR-181c-5p) on a rat model of neuropathic pain induced by sciatic nerve chronic constriction injury (CCI). In this study NP model was established using the CCI method. NP levels were assessed using PWT and PWL. Microarray analysis and RT-PCR were used to determine the relative expression of miR-181c-5p. MSC-derived exosomes were extracted using the total exosome isolation reagent characterized by WB and NTA. MiR-181c-5p was loading into Exos using electroporation. The inflammation response in microglia cells and CCI rats were assessed by ELISA assay respectively. Our study demonstrates that miR-181c-5p expression was obviously decreased in a time-dependent manner in CCI rats. MiR-181c-5p was effectively electroporated and highly detected in MSC-derived Exos. ExomiR-181c-5p internalized by microglia cells and inhibit the secretion of inflammation factors. ExomiR-181c-5p intrathecal administration alleviated neuropathic pain and neuroinflammation response in CCI rats. Taken together, ExomiR-181c-5p alleviated CCI-induced NP by inhibiting neuropathic inflammation. ExomiR-181c-5p may be a valid alternative for the treatment of neuropathic pain and has vast potential for future development.

Study on the Mechanism of Electroacupuncture in the Treatment of Neuropathic Pain Based on Vesicular Glutamate Transporter 2/Toll Like Receptor 4 Signal Pathway

To explore the analgesic effect of electro-acupuncture on neuropathic pain model rats and to clarify the mechanism of electro-acupuncture analgesia from the perspective of vesicular glutamate transporter 2/toll like receptor 4 signal pathway. A total of 30 specific pathogen free grade adult male Sprague-Dawley rats were selected. The rats were randomly divided into model group (chronic constriction injury), sham operation group, electro-acupuncture group, non-electro-acupuncture group (sham electro-acupuncture), Chicago sky blue 6B group and lipopolysaccharide group, with 5 rats in each group. The neuropathic pain model of chronic constriction injury of sciatic nerve was established. The “Huantiao” and “Yanglingquan” points were selected for electroacupuncture stimulation for 14 d. The mechanical foot contraction threshold, thermal pain, vesicular glutamate transporter 2/toll like receptor 4 signal pathway protein levels and inflammatory factor level of rats in each group were compared and analyzed. Compared with the sham operation group, the mechanical pain and thermal pain thresholds of the model group decreased significantly (p<0.01), the unilateral levels of vesicular glutamate transporter 2 and toll like receptor 4 and the release of interalukin-6 in the spinal cord increased significantly (p<0.01), while interalukin-10 decreased significantly (p<0.01); compared with the model group, electroacupuncture and intrathecal injection of Chicago sky blue 6B could significantly increase the pain threshold of neuropathic pain rats (p<0.01), reduce the release of interalukin-6 and the level of vesicular glutamate transporter 2 protein in the spinal cord and increase the level of interlukin-10; intrathecal injection of lipopolysaccharide decreased the pain threshold in rats (p<0.01). Electro-acupuncture can alleviate the pain sensitivity of neuropathic pain rats and its occurrence and development is closely related to the activation of vesicular glutamate transporter 2/toll like receptor 4 signal pathway in the spinal dorsal horn.

Jmjd3 Mediates Neuropathic Pain by Inducing Macrophage Infiltration and Activation in Lumbar Spinal Stenosis Animal Model.

Lumbar spinal stenosis (LSS) is a major cause of chronic neuropathic back and/or leg pain. Recently, we demonstrated that a significant number of macrophages infiltrated into the cauda equina after compression injury, causing neuroinflammation, and consequently mediating neuropathic pain development and/or maintenance. However, the molecular mechanisms underlying macrophage infiltration and activation have not been elucidated. Here, we demonstrated the critical role of histone H3K27 demethylase Jmjd3 in blood-nerve barrier dysfunction following macrophage infiltration and activation in LSS rats. The LSS rat model was induced by cauda equina compression using a silicone block within the epidural spaces of the L5-L6 vertebrae with neuropathic pain developing 4 weeks after compression. We found that Jmjd3 was induced in the blood vessels and infiltrated macrophages in a rat model of neuropathic pain. The blood-nerve barrier permeability in the cauda equina was increased after compression and significantly attenuated by the Jmjd3 demethylase inhibitor, GSK-J4. GSK-J4 also inhibited the expression and activation of MMP-2 and MMP-9 and significantly alleviated the loss of tight junction proteins and macrophage infiltration. Furthermore, the activation of a macrophage cell line, RAW 264.7, by LPS was significantly alleviated by GSK-J4. Finally, GSK-J4 and a potential Jmjd3 inhibitor, gallic acid, significantly inhibited mechanical allodynia in LSS rats. Thus, our findings suggest that Jmjd3 mediates neuropathic pain development and maintenance by inducing macrophage infiltration and activation after cauda equina compression and thus may serve as a potential therapeutic target for LSS-induced neuropathic pain.