Mechanical Withdrawal Threshold Research Articles

The Role and Mechanism of Spinal NF-κB-CXCL1/CXCR2 in Rats with Nucleus Pulposus-induced Radicular Pain.

Experimental study of the role and mechanism of spinal NFκB-CXCL1/CXCR2 in rats with nucleus pulposus-induced radicular pain. This study investigated the role and mechanism of spinal NFκB-CXCL1/CXCR2 in autologous nucleus pulposus-induced pain behavior in rats and to clarify the involvement and regulation of spinal NFκB as an upstream molecule of CXCL1 in autologous nucleus pulposus-induced radicular pain in rats. The inflammatory response of nerve roots is an important mechanism for the occurrence of chronic pain. NFκB-CXCL1/CXCR2 pathway plays an important role in the development of radicular pain, but its regulatory mechanism in the model of radicular pain induced by autologous nucleus pulposus is still unclear. We established a rat model of autologous medullary nucleus transplantation. We observed and recorded the changes in 50% mechanical withdrawal threshold and thermal withdrawal latency before and after the administration of CXCL1-neutralizing antibodies, CXCR2 inhibitor, and NFκB inhibitor in each group of rats and evaluated the expression of NFκB, CXCL1, and CXCR2 in the spinal dorsal horn using immunofluorescence and Western blot. To compare differences between groups in behavioral testing, analysis of variance was employed. Dunnett's method was used to compare differences at different time points within a group and between different groups at the same time point. A comparison of the relative concentration of protein, relative concentration of mRNA, and semiquantitative data from immunofluorescence staining was conducted utilizing one-way ANOVA and Dunnett's pairwise comparison. Autologous nucleus pulposus transplantation can induce radicular pain in rats and upregulate the expression of CXCL1, CXCR2, and NFκB in the spinal cord. CXCL1 is co-expressed with astrocytes, CXCR2 with neurons, and NFκB with both astrocytes and neurons. The application of CXCL1 neutralizing antibodies, CXCR2 inhibitors, and NFκB inhibitors can alleviate pain hypersensitivity induced by autologous nucleus pulposus transplantation in rats. Inhibitors of NFκB could downregulate the expression of CXCL1 and CXCR2. We found that spinal NFκB is involved in NP-induced radicular pain in rats through the activation of CXCL1/CXCR2, enriching the mechanism of medullary-derived radicular pain and providing a possible new target and theoretical basis for the development of more effective anti-inflammatory and analgesic drugs for patients with chronic pain following LDH.

Effects of Intrathecal Ketamine on Cerebrospinal Fluid Levels of Brain-Derived Neurotrophic Factor and Mechanical Allodynia in a Rat Model of Mild Traumatic Brain Injury.

BACKGROUND Ketamine, a compelling candidate for neuropathic pain management, has attracted interest for its potential to elevate brain-derived neurotrophic factor (BDNF) levels. We aimed to assess the effects of intrathecally administered ketamine on the cerebrospinal fluid (CSF) levels of BDNF(c-BDNF) and allodynia in a rat model of traumatic brain injury (TBI). MATERIAL AND METHODS Forty-five rats were divided into 3 groups: sham operation (Group S), untreated TBI (Group T), and ketamine-treated TBI (Group K), with 15 rats in each group. Rats were anesthetized, and their skulls were secured in a stereotactic frame before undergoing craniotomy. A controlled cortical impact (CCI) was induced, followed by injection of ketamine (3.41 µg/g) into the CSF in Group K. In Group T, no drug was injected after CCI delivery. On postoperative days (POD) 1, 7, and 14, the 50% mechanical withdrawal threshold (50% MWT) and c-BDNF levels were assessed. RESULTS Groups T and K exhibited a significantly lower 50% MWT than Group S on POD 1(6.6 [5.7, 8.7] g, 10.0 [6.8, 11.6] g, and 18.7 [11.6, 18.7] g, respectively; P<0.001). The c-BDNF levels in Group K were significantly higher than those in Groups S and T on POD 1 (18.9 [16.1, 23.0] pg/ml, 7.3 [6.0, 8.8] pg/ml, and 11.0 [10.6, 12.3] pg/ml, respectively; P=0.006). CONCLUSIONS Intrathecal ketamine administration did not exhibit anti-allodynic effects following mild TBI. c-BDNF level is a promising potential indicator for predicting the expression of allodynia after mild TBI.

Effect of metformin nanoemulsion through AMP-activated protein kinase (AMPK)-mediated autophagy on diabetic neuropathy

Metformin (MET) is the preferred first-line treatment for patients with type 2 diabetes. However, the mechanism of diabetic peripheral neuropathy (DPN) is still unclear. To improve the oral utilization of metformin, a metformin nanoemulsion (MET-NE) was prepared to investigate its effects on DPN and its underlying mechanism. In this study, a DPN model was established in Wistar rats induced by streptozotocin (STZ). The diabetic rats were randomly divided into four groups: the diabetic model group (DM group), the metformin tablet-treated group (MET group), the metformin nanoemulsion-treated group (MET-NE group), and a normal control group consisting of five normal Wistar rats. All groups were administered the treatment orally for a period of 10 weeks. The findings of the study demonstrated that both MET and MET-NE significantly reduced blood glucose levels, glycated serum protein levels, food intake, and water intake in DM rats. It was also observed that MET-NE was more effective than MET in reducing blood glucose levels. Additionally, both MET and MET-NE treatments significantly increased the motor nerve conduction velocity (MNCV), sensory nerve conduction velocity (SNCV), and sensory nerve action potential (SNAP) amplitude in DM rats. Furthermore, these treatments improved mechanical withdrawal threshold (MWT) and thermal withdrawal latency (TWL), resulting in a reduced sensitivity to pain stimuli. Moreover, both MET and MET-NE treatments promoted the phosphorylation of AMP-activated protein kinase (AMPK) and acetyl-CoA carboxylase (ACC) proteins, as well as increased the activity of autophagic proteins in the sciatic nerve. However, no significant differences were observed between MET and MET-NE treatments in terms of these effects. In conclusion, the MET-NE demonstrated a rapid decrease in blood glucose levels and improved glucose tolerance and metabolism, which was found to be superior to MET. Furthermore, MET-NE significantly improved the neurophysiological function and sciatic nerve pain threshold in DPN rats. These beneficial effects may be attributed to the regulation of AMPK-mediated autophagy by MET-NE.

Electroacupuncture at Sensitized Acupoints Relieves Somatic Referred Pain in Colitis Rats by Inhibiting Sympathetic-Sensory Coupling to Interfere with 5-HT Signaling Pathway.

To investigate whether electroacupuncture (EA) at sensitized acupoints could reduce sympathetic-sensory coupling (SSC) and neurogenic inflammatory response by interfering with 5-hydroxytryptamine (5-HT)ergic neural pathways to relieve colitis and somatic referred pain, and explore the underlying mechanisms. Rats were treated with 5% dextran sodium sulfate (DSS) solution for 7 days to establish a colitis model. Twelve rats were randomly divided into the control and model groups according to a random number table (n=6). According to the "Research on Rat Acupoint Atlas", sensitized acupoints and non-sensitized acupoints were determined. Rats were randomly divided into the control, model, Zusanli-EA (ST 36), Dachangshu-EA (BL 25), and Xinshu (BL 15) groups (n=6), as well as the control, model, EA, and EA + GR113808 (a 5-HT inhibitor) groups (n=6). The rats in the control group received no treatment. Acupuncture was administered on 2 days after modeling using the stimulation pavameters: 1 mA, 2 Hz, for 30 min, with sparse and dense waves, for 14 consecutive days. GR113808 was injected into the tail vein at 5 mg/kg before EA for 10 min for 7 consecutive days. Mechanical sensitivity was assessed with von Frey filaments. Body weight and disease activity index (DAI) scores of rats were determined. Hematoxylin and eosin staining was performed to observe colon histopathology. SSC was analyzed by immunofluorescence staining. Immunohistochemical staining was performed to detect 5-HT and substance P (SP) expressions. The calcitonin gene-related peptide (CGRP) in skin tissue and tyrosine hydroxylase (TH) protein levels in DRG were detected by Western blot. The levels of hyaluronic acid (HA), bradykinin (BK), prostaglandin I2 (PGI2) in skin tissue, 5-HT, tryptophan hydroxylase 1 (TPH1), serotonin transporters (SERT), 5-HT 3 receptor (5-HT3R), and 5-HT 4 receptor (5-HT4R) in colon tissue were measured by enzyme-linked immunosorbent assay (ELISA). BL 25 and ST 36 acupoints were determined as sensitized acupoints, and BL 15 acupoint was used as a non-sensitized acupoint. EA at sensitized acupoints improved the DAI score, increased mechanical withdrawal thresholds, and alleviated colonic pathological damage of rats. EA at sensitized acupoints reduced SSC structures and decreased TH and CGRP expression levels (P<0.05). Furthermore, EA at sensitized acupoints reduced BK, PGI2, 5-HT, 5-HT3R and TPH1 levels, and increased HA, 5-HT4R and SERT levels in colitis rats (P<0.05). GR113808 treatment diminished the protective effect of EA at sensitized acupoints in colitis rats (P<0.05). EA at sensitized acupoints alleviated DSS-induced somatic referred pain in colitis rats by interfering with 5-HTergic neural pathway, and reducing SSC inflammatory response.

Liquid Crystal Polymer-Based Miniaturized Fully Implantable Deep Brain Stimulator.

A significant challenge in improving the deep brain stimulation (DBS) system is the miniaturization of the device, aiming to integrate both the stimulator and the electrode into a compact unit with a wireless charging capability to reduce invasiveness. We present a miniaturized, fully implantable, and battery-free DBS system designed for rats, using a liquid crystal polymer (LCP), a biocompatible and long-term reliable material. The system integrates the simulator circuit, the receiver coil, and a 20 mm long depth-type microelectrode array in a dome-shaped LCP package that is 13 mm in diameter and 5 mm in height. Wireless powering and control via an inductive link enable device miniaturization, allowing for full implantation and, thus, the free behavior of untethered animals. The eight-channel stimulation electrode array was microfabricated on an LCP substrate to form a multilayered system substrate, which was monolithically encapsulated by a domed LCP lid using a specialized spot-welding process. The device functionality was validated via an in vivo animal experiment using a neuropathic pain model in rats. This experiment demonstrated an increase in the mechanical withdrawal threshold of the rats with microelectrical stimulation delivered using the fully implanted device, highlighting the effectiveness of the system.

Electroacupuncture promotes synaptic plasticity in rats with chronic inflammatory pain-related depression by upregulating BDNF/TrkB/CREB signaling pathway.

Chronic inflammatory pain (CIP) frequently coincides with depression among patients. The onset and development of pain and depression are associated with altered neural synaptic plasticity. Electroacupuncture (EA) can effectively relieve CIP and depression. However, the underlying mechanisms have not been fully illustrated. To explore whether EA can relieve CIP and depression by regulating hippocampal synaptic plasticity, and the present study offers foundational evidence for the efficacy of EA in treating CIP-related depression (CIPD). Rats were divided into four groups: 0.9% normal saline group, complete Freund's adjuvant (CFA) group, CFA+duloxetine group, and CFA+EA group. Pain hypersensitivity was detected by mechanical withdrawal threshold and thermal paw withdrawal latency, and the depression level was gauged using the open field test, the sucrose preference test, and the forced swimming test. The morphology of the hippocampal neurons was observed using Nissl staining. The protein expression levels of synuclein (Syn), postsynaptic density protein-95 (PSD-95), brain-derived neurotrophic factors (BDNFs), tyrosine-protein kinase B (TrKB), p-TrkB, cAMP response element binding protein (CREB), and p-CREB were measured by western blotting and immunofluorescence staining. BDNF and TrkB mRNA expression were detected using quantitative real-time polymerase chain reaction (PCR) (qRT-PCR). The content of 5-hydroxytryptamine (5-HT) and γ-aminobutyric acid (GABA) was detected using enzyme-linked immunosorbent assay, and the glutamic acid (Glu) content was determined using the ultraviolet colorimetry method. The hippocampal neuron ultrastructure was observed using transmission electron microscopy. EA could alleviate CIP and related depressive behaviors as well as protect the hippocampal neuronal structure from damage and regulate 5-HT/GABA/Glu levels in the hippocampus. Additionally, EA could significantly increase the expression of synapse-associated proteins such as PSD-95 and Syn by activating the BDNF/TrKB/CREB signaling pathway. EA improves pain and depressive behaviors in CIPD rats, and the mechanism may be related to synaptic plasticity mediated by the BDNF/TrKB/CREB signaling pathway.

Medication overuse headache is associated with elevated lipopolysaccharide binding protein and pro-inflammatory molecules in the bloodstream

ObjectiveMedication overuse headache (MOH) is a secondary headache that accompanies chronic migraine. Nonsteroidal anti-inflammatory drugs (NSAIDs) are the most frequently used analgesics worldwide and they are known to induce leaky gut. In this study, we aimed to investigate whether NSAID induced MOH is associated with altered circulating lipopolysaccharide binding protein (LBP) levels and inflammatory molecules.Materials and methodsPiroxicam (10 mg/kg/day, po) for 5 weeks was used to induce MOH in female Sprague Dawley rats. Pain behavior was evaluated by periorbital withdrawal thresholds, head-face grooming, freezing, and head shake behavior. Serum samples and brain tissues were collected to measure circulating LBP, tight junction protein occludin, adherens junction protein vascular endothelial (VE)-cadherin, calcitonin gene-related peptide (CGRP), IL-6 levels and brain high mobility group box-1 (HMGB1) and IL-17 levels.ResultsChronic piroxicam exposure resulted in decreased periorbital mechanical withdrawal thresholds, increased head-face grooming, freezing, and head shake behavior compared to vehicle administration. Serum LBP, CGRP, IL-6, IL-17, occludin, VE-cadherin levels and brain IL-17 and HMGB1 levels were significantly higher in piroxicam group compared to controls. Serum LBP was positively correlated with occludin (r = 0.611), VE-cadherin (r = 0.588), CGRP (r = 0.706), HMGB1 (r = 0.618) and head shakes (r = 0.921), and negatively correlated with periorbital mechanical withdrawal thresholds (r = -0.740).ConclusionElevated serum LBP, VE-cadherin and occludin levels indicating disrupted intestinal barrier function and leakage of LPS into the systemic circulation were shown in female rats with MOH. LPS induced low-grade inflammation and elevated nociceptive and/or pro-inflammatory molecules such as HMGB1, IL-6, IL-17 and CGRP may play a role in the development and maintenance of MOH. Interference with leaky gut and pro-inflammatory nociceptive molecules could also be a target for sustained management of MOH.

Upregulation of Calhm2 in the anterior cingulate cortex contributes to the maintenance of bilateral mechanical allodynia and comorbid anxiety symptoms in inflammatory pain conditions

Peripheral inflammation-induced chronic pain tends to evoke concomitant anxiety disorders. It's common knowledge that the anterior cingulate cortex (ACC) plays a vital role in maintaining pain modulation and negative emotions. However, the potential mechanisms of chronic inflammation pain and pain-related anxiety remain elusive. Here, it was reported that injecting complete Freund’s adjuvant (CFA) unilaterally resulted in bilateral mechanical allodynia and anxiety-like symptoms in mice via behavioral tests. In addition, CFA induced the bilateral upregulation and activation of calcium homeostasis modulator 2 (Calhm2) in ACC pyramidal neurons by quantitative analysis and double immunofluorescence staining. The knockdown of Calhm2 in the bilateral ACC by a lentiviral vector harboring ribonucleic acid (RNA) interference sequence reversed CFA-induced pain behaviors and neuronal sensitization. Furthermore, the modulating of ACC pyramidal neuronal activities via a designer receptor exclusively activated by designer drugs (DREADD)-hM4D(Gi) greatly changed Calhm2 expression, mechanical paw withdrawal thresholds (PWTs) and comorbid anxiety symptoms. Moreover, it was found that Calhm2 regulates inflammation pain promoting the upregulation of N-methyl-D-aspartic acid (NMDA) receptor 2B (NR2B) subunits. Calhm2 knockdown in ACC exhibited a significant decrease in NR2B expression. These results demonstrated that Calhm2 in ACC pyramidal neurons modulates chronic inflammation pain and pain-related anxiety symptoms, which provides a novel underlying mechanism for the development of inflammation pain.

Electroacupuncture exerts prolonged analgesic and neuroprotective effects in a persistent dental pain model induced by multiple dental pulp injuries: GABAergic interneurons-astrocytes interaction.

Pain within the trigeminal system, particularly dental pain, is poorly understood. This study aimed to determine whether single or multiple dental pulp injuries induce persistent pain, its association with trigeminal central nociceptive pathways and whether electroacupuncture (EA) provides prolonged analgesic and neuroprotective effects in a persistent dental pain model. Models of single dental pulp injury (SDPI) and multiple dental pulp injuries (MDPI) were used to induce trigeminal neuropathic pain. The signs of dental pain-related behavior were assessed using the mechanical head withdrawal threshold (HWT). Immunofluorescence and western blot protocols were used to monitor astrocyte activation, changes in apoptosis-related proteins, and GABAergic interneuron plasticity. SDPI mice exhibited an initial marked decrease in HWT from days one to 14, followed by progressive recovery from days 21 to 42. From days 49 to 70, the HWT increased and returned to the control values. In contrast, MDPI mice showed a persistent decrease in HWT from days one to 70. MDPI increased glial fibrillary acidic protein (GFAP) and decreased glutamine synthetase (GS) and glutamate transporter-1 (GLT1) expression in the Vi/Vc transition zone of the brainstem on day 70, whereas no changes in astrocytic markers were observed on day 70 after SDPI. Increased expression of cleaved cysteine-aspartic protease-3 (cleaved caspase-3) and Bcl-2-associated X protein (Bax), along with decreased B-cell lymphoma/leukemia 2 (Bcl-2), were observed at day 70 after MDPI but not after SDPI. The downregulation of glutamic acid decarboxylase (GAD65) expression was observed on day 70 only after MDPI. The effects of MDPI-induced lower HWT from days one to 70 were attenuated by 12 sessions of EA treatment (days one to 21 after MDPI). Changes in astrocytic GFAP, GS, and GLT-1, along with cleaved caspase-3, Bax, Bcl-2, and GAD65 expression observed 70 days after MDPI, were reversed by EA treatment. The results suggest that persistent dental pain in mice was induced by MDPI but not by SDPI. This effect was associated with trigeminal GABAergic interneuron plasticity along with morphological and functional changes in astrocytes. EA exerts prolonged analgesic and neuroprotective effects that might be associated with the modulation of neuron-glia crosstalk mechanisms.

BMP7 alleviates trigeminal neuralgia by reducing oligodendrocyte apoptosis and demyelination

BackgroundBMP7 has been shown to have neuroprotective effects and to alleviate demyelination. However, its role in trigeminal neuralgia (TN) has not been well investigated. The current study aims to determine whether BMP7 plays a role in demyelination, its effects on pain behaviors and mechanism of action in rats with TN.MethodsWe used an infraorbital-nerve chronic-constriction injury (ION-CCI) to establish a rat model of TN. Adeno-associated viruses (AAVs) were injected into the rats to upregulate or downregulate BMP7. The mechanical withdrawal thresholds (MWT) of the injured rats were detected using Von Frey filaments. The changes in expression levels of BMP7 and oligodendrocyte (OL) markers were examined by western blotting, quantitative real-time PCR, immunofluorescence, and transmission electron microscopy.ResultsThe ION-CCI induced mechanical allodynia, demyelination, and loss of OLs with a reduction of BMP7. Short-hairpin RNA (shRNA)-BMP7 that inhibited BMP7 expression also caused mechanical allodynia, demyelination, and loss of OLs, and its mechanism may be OL apoptosis. Overexpressing BMP7 in the trigeminal spinal subnucleus caudalis(VC) with AAV-BMP7 relieved all three phenotypes induced by the CCI, and its mechanism may be alleviating OLs apoptosis. Two signal pathways associated with apoptosis, STAT3 and p65, were significantly downregulated in the VC after CCI and rescued by BMP7 overexpression.ConclusionBMP7 can alleviate TN by reducing OLs apoptosis and subsequent demyelination. The mechanism behind this protection could be BMP7-mediated activation of the STAT3 and NF-κB/p65 signaling pathway and subsequent decrease in OL apoptosis. Importantly, our study presents clear evidence in support of BMP7 as a possible therapeutic target for the treatment of TN.

Linarin attenuates oxaliplatin-induced neuropathic pain by inhibiting NF-κB/NLRP3 signaling pathway

Purpose: To assess the therapeutic effects of linarin on chemotherapy-induced peripheral neuropathy (CINP) in rats.Methods: A CINP rat model was established using oxaliplatin. The rats were divided into control, CINP, and two linarin treatment groups (20 mg/kg/day and 40 mg/kg/day). Observations were made at various time points, assessing weight gain, mechanical withdrawal thresholds, cold allodynia response, and thermal pain sensitivity. Additionally, the expression levels of various inflammatory factors (IL-1β, IL-6, IL-10, and IL-17), proteins related to glial and neuronal activation (IBA-1, GFAP, c-fos), and proteins linked to NF-κB/NLRP3 signaling (ASC, caspase-1, p65, and p-p65) were evaluated in rat spinal cord tissue.Results: Linarin treatment resulted in improved weight gain, mechanical threshold, decreased withdrawal response, and enhanced paw withdrawal latency (p &lt; 0.001) compared to the CINP group. These improvements or mitigations were more pronounced in the 40 mg/kg/day linarin group. Linarin inhibited the expression of inflammatory factors IL-1β, IL-6, and IL-17 (p &lt; 0.001) but enhanced IL-10 expression (p &lt; 0.001). The activation of microglia, astrocytes, and neurons, as indicated by IBA-1, GFAP, and c-fos (p &lt; 0.001) proteins, was significantly reduced with linarin, especially at the higher dose. Linarin also suppressed the expression of ASC, caspase-1, p65, and p-p65 (p &lt; 0.001) proteins, associated with the NF-κB/NLRP3 signaling pathway.Conclusion: Our study indicates that linarin may serve as a potential therapeutic agent for managing CINP. The beneficial effects of linarin are likely mediated through its immunomodulatory effects and the inhibition of the NF-κB/NLRP3 signaling pathway. Further research is needed to confirm these findings in clinical settings.

Electroacupuncture stimulating Zhongji (CV3), Guanyuan (CV4), and bilateral Dahe (KI12) attenuates inflammation in rats with chronic nonbacterial prostatitis induced by estradiol through inhibiting toll-like receptor 4 pathway.

To investigate the anti-inflammatory effect of electroacupuncture (EA) on chronic prostatitis/chronic pelvic pain syndrome (CP/CPPS), also known as chronic nonbacterial prostatitis (CNP), and explore its underlying mechanism. A CNP rat was established by surgical castration combined with 17-β estradiol injection in male Sprague-Dawley rats for thirty consecutive days. The CNP rats received EA treatment once a day for eight days. Chronic pelvic pain was evaluated by mechanical withdrawal threshold measurement. The histological change was assessed by hematoxylin-eosin staining. The inflammatory cytokines in prostates were determined by enzyme-linked immunosorbent assays. The expressions of toll-like receptor 4 (TLR4), myeloid differentiation factor 88 (MyD88), inhibitors of kappa-B alpha (IκBα), and nuclear factor-kappa B (NF-κB) were detected by Western blotting. The nuclear translocation of NF-κB and the location of TLR4 were observed with immunofluorescence staining. The results showed that EA decreased the prostate index, upregulated the mechanical withdrawal threshold, restored the histomorphology of the prostate, reduced the inflammatory factor levels, inhibited NF-κB p65 nuclear translocation, and downregulated the expression levels of critical proteins involved in the TLR4/NF-κB signaling pathway in prostates. Our findings suggested that EA could relieve pelvic pain and attenuate prostatic inflammation in estradiol-induced CNP rats. The underlying mechanism may be related to the inhibition of the TLR4/NF-κB signaling pathway.

Morphine induces HADHA succinylation, while HADHA desuccinylation alleviates morphine tolerance by influencing autophagy.

Morphine tolerance is an important factor in unsatisfactory analgesia. HADHA is a crucial enzyme in fatty acid β-oxidation. In this study, we investigated the potential significance of HADHA in a mechanism that might cause morphine tolerance related to functional changes in energy metabolism and further explored the effect of HADHA desuccinylation on morphine tolerance. Rats received daily intrathecal injections of 10 µg of morphine for a duration of 7 consecutive days, and pain thresholds were measured using the mechanical withdrawal threshold (MWT) and thermal tail flick latency (TFL) tests. µ-Opioid receptor (MOR), LC3-I/II, and P62 expression and HADHA succinylation were assessed. HADHA succinylation was analyzed by liquid chromatography-tandem mass spectrometry (LC‒MS/MS) and parallel reaction monitoring (PRM). Morphine influenced the LC3II/LC3I ratio and P62 expression level, which are crucial indicators of autophagy, and stimulated HADHA succinylation. Additionally, HADHA was selectively bound by the desuccinylase SIRT5, and SIRT5 overexpression decreased HADHA succinylation, reduced P62 expression, and alleviated morphine tolerance.

Duhuo Jisheng decoction alleviates neuroinflammation and neuropathic pain by suppressing microglial M1 polarization: a network pharmacology research

BackgroundNeuropathic pain (NP) is the most prevalent form of chronic pain resulting from nerve damage or injury. Despite the widespread use of Duhuo Jisheng decoction (DHJSD) in traditional Chinese medicine (TCM) to treat chronic pain, the mechanism underlying its analgesic action remains unclear.MethodsUsing network pharmacology, we obtained DHJSD and NP-related target information from public databases to construct protein–protein interactions (PPI) and compound-target networks based on common target genes. These networks were further analyzed using gene ontology (GO) and Kyoto encyclopedia of genes and genomes (KEGG). The interaction between molecules was verified through molecular docking using AutoDock Tools software. Additionally, we treated a chronic constriction injury (CCI) rat model with DHJSD and determined the mechanical withdrawal threshold (MWT). We used an enzyme-linked immunosorbent assay kit to determine the levels of inflammatory cytokines. Furthermore, qRT-PCR was employed to analyze ACHE, NOS2, MAPK3, PTGS2, AKT1, and PPARG mRNA expression, and immunofluorescence was used to evaluate changes in microglia.ResultsOur screening of compounds and targets identified 252 potential targets of DHJSD associated with NP. PPI analysis, along with GO and KEGG analyses, revealed that the potential mechanism of DHJSD in NP treatment may be related to inflammatory reactions, the IL-17 signaling pathway, MAP kinase activity, and endocrine activity. Based on molecular docking, the core target showed significant affinity for DHJSD's active components. Moreover, DHJSD treatment repaired the CCI-induced inflammatory reaction in the spinal cord while regulating the expression of ACHE, NOS2, MAPK3, PTGS2, AKT1, and PPARG mRNA. Immunofluorescence results indicated that the active components of DHJSD may regulate microglial M1 polarization to improve neuroinflammation, PPARG may have been involved in the process.ConclusionThe multi-component, multi-target, and multi-pathway actions of DHJSD provide new insights into its therapeutic mechanism in NP.

Repeated closed-head mild traumatic brain injury-induced inflammation is associated with nociceptive sensitization.

Individuals who have experienced mild traumatic brain injuries (mTBIs) suffer from several comorbidities, including chronic pain. Despite extensive studies investigating the underlying mechanisms of mTBI-associated chronic pain, the role of inflammation in long-term pain after mTBIs is not fully elucidated. Given the shifting dynamics of inflammation, it is important to understand the spatial-longitudinal changes in inflammatory processes following mTBIs and their effects on TBI-related pain. We utilized a recently developed transgenic caspase-1 luciferase reporter mouse model to monitor caspase-1 activation through a thinned skull window in the in vivo setting following three closed-head mTBI events. Organotypic coronal brain slice cultures and acutely dissociated dorsal root ganglion (DRG) cells provided tissue-relevant context of inflammation signal. Mechanical allodynia was assessed by mechanical withdrawal threshold to von Frey and thermal hyperalgesia withdrawal latency to radiant heat. Mouse grimace scale (MGS) was used to detect spontaneous or non-evoked pain. In some experiments, mice were prophylactically treated with MCC950, a potent small molecule inhibitor of NLRP3 inflammasome assembly to inhibit injury-induced inflammatory signaling. Bioluminescence spatiotemporal dynamics were quantified in the head and hind paws, and caspase-1 activation was confirmed by immunoblot. Immunofluorescence staining was used to monitor the progression of astrogliosis and microglial activation in ex vivo brain tissue following repetitive closed-head mTBIs. Mice with repetitive closed-head mTBIs exhibited significant increases of the bioluminescence signals within the brain and paws in vivo for at least one week after each injury. Consistently, immunoblotting and immunofluorescence experiments confirmed that mTBIs led to caspase-1 activation, astrogliosis, and microgliosis. Persistent changes in MGS and hind paw withdrawal thresholds, indicative of pain states, were observed post-injury in the same mTBI animals in vivo. We also observed enhanced inflammatory responses in ex vivo brain slice preparations and DRG for at least 3days following mTBIs. In vivo treatment with MCC950 significantly reduced caspase-1 activation-associated bioluminescent signals in vivo and decreased stimulus-evoked and non-stimulus evoked nociception. Our findings suggest that the inflammatory states in the brain and peripheral nervous system following repeated mTBIs are coincidental with the development of nociceptive sensitization, and that these events can be significantly reduced by inhibition of NLRP3 inflammasome activation.

Vacuolar-ATPase-mediated muscle acidification caused muscular mechanical nociceptive hypersensitivity after chronic stress in rats, which involved extracellular matrix proteoglycan and ASIC3

Although widespread pain, such as fibromyalgia, is considered to have a central cause, peripheral input is important. We used a rat repeated cold stress (RCS) model with many characteristics common to fibromyalgia and studied the possible involvement of decreased muscle pH in muscle mechanical hyperalgesia. After a 5-day RCS, the muscle pH and the muscular mechanical withdrawal threshold (MMWT) decreased significantly. Subcutaneously injected specific inhibitor of vacuolar ATPase (V-ATPase), bafilomycin A1, reversed both changes almost completely. It also reversed the increased mechanical response of muscle thin-fibre afferents after RCS. These results show that V-ATPase activation caused muscle pH drop, which led to mechanical hypersensitivity after RCS. Since extracellular matrix proteoglycan and acid sensitive ion channels (TRPV1 and ASIC3) have been considered as possible mechanisms for sensitizing/activating nociceptors by protons, we investigated their involvement. Manipulating the extracellular matrix proteoglycan with chondroitin sulfate and chondroitinase ABC reversed the MMWT decrease after RCS, supporting the involvement of the extracellular mechanism. Inhibiting ASIC3, but not TRPV1, reversed the decreased MMWT after RCS, and ASIC3 mRNA and protein in the dorsal root ganglia were upregulated, indicating ASIC3 involvement. These findings suggest that extracellular mechanism and ASIC3 play essential roles in proton-induced mechanical hyperalgesia after RCS.

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

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

Comparisons of the analgesic effect of different pulsed radiofrequency targets in SNI-induced neuropathic pain.

An injury of the peripheral nerve may lead to neuropathic pain, which could be treated with pulsed radiofrequency to the dorsal root ganglion (DRG) or peripheral nerve [the nerve trunk (NT) or proximal to the injury site (NI)]. However, it is not clear whether there is any difference in analgesic effect or maintenance among the three targets. PRF was applied to the ipsilateral L5 DRG, peripheral nerve (NT or NI) 5 days after spared nerve injury (SNI). Triptolide (10 µg/kg) or vehicle was intrathecally administered 5 days after SNI for 3 days. Mechanical withdrawal thresholds were tested after treatment at different time points. Furthermore, microglia and the P2X7 receptor (P2X7R) in the ipsilateral spinal cord were measured with immunofluorescence and western blotting, respectively. PRF + NI exerted a more remarkable analgesic effect than PRF + DRG and PRF + NT at the early stage, but PRF + DRG had a stronger analgesic effect than PRF + NI and PRF + NT at the end of our study. In addition, PRF + DRG showed no significant difference from intrathecal administration of triptolide. Moreover, SNI-induced microglia activation and upregulation of P2X7R in spinal dorsal horn could be markedly inhibited by PRF + DRG. The results suggest that the analgesic effect of PRF + DRG increased with time whereas the other two not and microglia and P2X7R in the ipsilateral spinal dorsal horn may be involved in the process.

Electroacupuncture alleviates neuropathic pain caused by SNL by promoting M2 microglia polarization through PD-L1

As a common clinical disease, neuropathic pain is difficult to be cured with drugs. The occurrence and progression of pain is closely related to the response of spinal microglia. Aspartof the regulation of microglialactivity,PD-L1 playsacriticalrole. Loss of PD-L1 promoted the polarization of M1-like microglia. Increased expression of PD-L1 promoted M2-like polarization. Electroacupuncture has a significant analgesic effect in clinical practice, but its specific mechanism remains to be further explored. In this study, we verified the role of PD-L1 in EA analgesia and the underlying molecular mechanism through spinal nerve ligation (SNL) in rats and lipopolysaccharide (LPS)-treated BV2 microglial cells. Forbehavioralstudiesofrats,mechanical withdrawal threshold (MWT) and thermal withdrawal latency (TWL) were measured, and spinal cord neuros were examined under transmission electron microscopyto determine changes to their myelin structure. The expression levels of PD-L1 and M1/M2-specific markers in rat spinal cord and BV2 microglial cells were measured by enzyme-linked immunosorbent assay, flow cytometry, immunofluorescence staining and Western blot analysis. Our study showed that EA increased the pain threshold, reduced the destruction of myelin structure, promoted the expression of PD-L1 and PD-1, inhibited the MAPK signaling pathway, and promoted the conversion of microglial polarization from the M1 phenotype to the M2 phenotype in SNL rats. PD-L1 knockdown reversed these effects of EA. In addition, PD-L1 knockdown activated the MAPK signaling pathway, promoted microglial polarization to the M1 phenotype, decreased the expression of anti-inflammatory mediators and increased the expression of proinflammatory factors in LPS-stimulated BV2 microglial cells. Our results showed that EA may regulate the excitability of primary afferent neurons through PD-L1 and then inhibit the MAPK signaling pathway to promote the transformation of activated M1 microglia into M2 microglia, reduce inflammatory reactions, and finally achieve analgesic effects. A therapy targeting PD-L1 may be an effective strategy for treating neuropathic pain.

NETO2-GluK2 interaction contributes to postoperative pain hypersensitivity through inducing PKCγ activation and synaptic incorporation of AMPA receptor GluR1 subunits in rat dorsal horn

Important roles in the initiation and maintenance of postoperative pain are played by the functional control of kainate (KA) and α-amino-3-hydroxy-5-methyl-4-isoxazole propionate (AMPA) receptors in the rat dorsal horn (DH). However, the mechanisms underpinning the cross-talk between spinal KA and AMPA receptors in postoperative pain are poorly understood. We hypothesized that after the rat’s plantar incision, the synaptic incorporation of AMPA receptor GluR1 subunits in the DH ipsilateral to the incision would increase due to the interaction between GluK2 and neuropilin tolloid-like 2 (NETO2). Our findings showed that incision stimuli caused severe pain responses, as measured by cumulative pain scores. GluK2-NETO2 but not GluK2-NETO1interaction was upregulated in ipsilateral dorsal horn neurons (DHNs) at 6 h post-incision. At 6 h post-incision, NETO2 small interfering ribonucleic acid (siRNA) intrathecal pretreatment increased mechanical withdrawal thresholds to von Freys and decreased ipsilateral paw cumulative pain scores. Further, PKCγactivation and synaptic abundance of GluK2 and GluR1 subunits in the ipsilateral DH were decreased by intrathecal pretreatment with NETO2 siRNA at 6 h post-incision. In conclusion, our findings imply that GluK2-NETO2 interaction could trigger PKCγactivation and the synaptic incorporation of AMPA receptor GluR1 subunits in rat DHs, which in turn led to the enhanced pain hypersensitivity after surgery. It sheds light on the interplay between KA and AMPA receptors in DHNs, which is thought to contribute to postoperative pain.