OBJECTIVE: To investigate the effect of midazolam on pain in lumbar disc herniation model rats based on p38 MAPK signaling pathway. METHODS: Fifty SPF-grade Sprague-Dawley healthy rats, half male and half female, were selected and randomly divided into normal group, model group, and low-dose, medium-dose, high-dose groups. Model group and low-dose, medium-dose, high-dose groups were initially modeled for lumbar disc herniation. Intraperitoneal injection of saline was performed in rats of normal and model groups; and in the low-dose, medium-dose, and high-dose groups, intraperitoneal injection of midazolam was performed with doses of 30, 60, and 90 mg/kg, respectively. Interleukin-1β (IL-1β), tumor necrosis factor-α (TNF-α), 5-hydroxytryptamine (5-HT), β-endorphin (β-EP), substance P (SP), neuropeptide Y (NPY) were detected in the serum of rats by enzyme-linked immunoassay. The expression of p38 MAPK and matrix metalloproteinase-3(MMP-3) protein were detected by Western blot in the tissues of rats of each group. RESULTS: The levels of TNF-α, IL-1β and β-EP were higher and the level of 5-HT was lower in the model group than in the normal group(P<0.05);the levels of TNF-α, IL-1β and β-EP were lower and the level of 5-HT was higher in the low-dose, medium-dose and high-dose groups than in the model group(P<0.05). The levels of SP and NPY increased in the model group compared with the normal group (P<0.05) and the levels of SP and NPY decreased in the low-dose, medium-dose and high-dose groups compared with the model group (P<0.05). The expression of p38 MAPK and MMP-3 increased in the model group compared with the normal group (P<0.05); the expression of p38 MAPK and MMP-3 decreased in the low-dose, medium-dose and high-dose compared with the model group(P<0.05). CONCLUSION Midazolam may ameliorate the immune inflammatory response in rats with a model of lumbar disc herniation, possibly regulated through the p38MAPK signaling pathway.
Rats ; Male ; Female ; Animals ; Intervertebral Disc Displacement/pathology* ; Rats, Sprague-Dawley ; Matrix Metalloproteinase 3/metabolism* ; Midazolam ; Tumor Necrosis Factor-alpha/metabolism* ; Serotonin/metabolism* ; MAP Kinase Signaling System/physiology* ; Pain ; p38 Mitogen-Activated Protein Kinases/metabolism*

Post-amputation pain causes great suffering to amputees, but still no effective drugs are available due to its elusive mechanisms. Our previous clinical studies found that surgical removal or radiofrequency treatment of the neuroma at the axotomized nerve stump effectively relieves the phantom pain afflicting patients after amputation. This indicated an essential role of the residual nerve stump in the formation of chronic post-amputation pain (CPAP). However, the molecular mechanism by which the residual nerve stump or neuroma is involved and regulates CPAP is still a mystery. In this study, we found that nociceptors expressed the mechanosensitive ion channel TMEM63A and macrophages infiltrated into the dorsal root ganglion (DRG) neurons worked synergistically to promote CPAP. Histology and qRT-PCR showed that TMEM63A was mainly expressed in mechanical pain-producing non-peptidergic nociceptors in the DRG, and the expression of TMEM63A increased significantly both in the neuroma from amputated patients and the DRG in a mouse model of tibial nerve transfer (TNT). Behavioral tests showed that the mechanical, heat, and cold sensitivity were not affected in the Tmem63a^-/- mice in the naïve state, suggesting the basal pain was not affected. In the inflammatory and post-amputation state, the mechanical allodynia but not the heat hyperalgesia or cold allodynia was significantly decreased in Tmem63a^-/- mice. Further study showed that there was severe neuronal injury and macrophage infiltration in the DRG, tibial nerve, residual stump, and the neuroma-like structure of the TNT mouse model, Consistent with this, expression of the pro-inflammatory cytokines TNF-α, IL-6, and IL-1β all increased dramatically in the DRG. Interestingly, the deletion of Tmem63a significantly reduced the macrophage infiltration in the DRG but not in the tibial nerve stump. Furthermore, the ablation of macrophages significantly reduced both the expression of Tmem63a and the mechanical allodynia in the TNT mouse model, indicating an interaction between nociceptors and macrophages, and that these two factors gang up together to regulate the formation of CPAP. This provides a new insight into the mechanisms underlying CPAP and potential drug targets its treatment.
Animals ; Mice ; Amputation, Surgical ; Chronic Pain/pathology* ; Disease Models, Animal ; Ganglia, Spinal/pathology* ; Hyperalgesia/etiology* ; Ion Channels/metabolism* ; Macrophages ; Neuroma/pathology*

Humans ; Sodium-Calcium Exchanger ; Carrier Proteins ; Pain ; Calcium/metabolism*

Although sympathetic blockade is clinically used to treat pain, the underlying mechanisms remain unclear. We developed a localized microsympathectomy (mSYMPX), by cutting the grey rami entering the spinal nerves near the rodent lumbar dorsal root ganglia (DRG). In a chemotherapy-induced peripheral neuropathy model, mSYMPX attenuated pain behaviors via DRG macrophages and the anti-inflammatory actions of transforming growth factor-β (TGF-β) and its receptor TGF-βR1. Here, we examined the role of TGF-β in sympathetic-mediated radiculopathy produced by local inflammation of the DRG (LID). Mice showed mechanical hypersensitivity and transcriptional and protein upregulation of TGF-β1 and TGF-βR1 three days after LID. Microsympathectomy prevented mechanical hypersensitivity and further upregulated Tgfb1 and Tgfbr1. Intrathecal delivery of TGF-β1 rapidly relieved the LID-induced mechanical hypersensitivity, and TGF-βR1 antagonists rapidly unmasked the mechanical hypersensitivity after LID+mSYMPX. In situ hybridization showed that Tgfb1 was largely expressed in DRG macrophages, and Tgfbr1 in neurons. We suggest that TGF-β signaling is a general underlying mechanism of local sympathetic blockade.
Mice ; Animals ; Receptor, Transforming Growth Factor-beta Type I/metabolism* ; Transforming Growth Factor beta/pharmacology* ; Transforming Growth Factor beta1/metabolism* ; Hyperalgesia/metabolism* ; Radiculopathy/metabolism* ; Pain/metabolism* ; Analgesics/pharmacology* ; Ganglia, Spinal/metabolism*

The Baimai Ointment with the effect of relaxing sinew and activating collaterals demonstrates a definite effect on Baimai disease with pain, spasm, stiffness and other symptoms, while the pharmacodynamic characteristics and mechanism of this agent remain unclear. In this study, a rat model of chronic compression of L4 dorsal root ganglion(CCD) was established by lumbar disc herniation, and the efficacy and mechanism of Baimai Ointment in the treatment of CCD were preliminarily explored by behavioral tests, side effect evaluation, network analysis, antagonist and molecular biology verification. The pharmacodynamic experiment indicated that Baimai Ointment significantly improved the pain thresholds(mechanical pain, thermal pain, and cold pain) and gait behavior of CCD model rats without causing tolerance or obvious toxic and side effects. Baimai Ointment inhibited the second-phase nociceptive response of mice in the formalin test, increased the hot plate threshold of normal mice, and down-regulated the expression of inflammatory cytokines in the spinal cord. Network analysis showed that Baimai Ointment had synergistic effect in the treatment of CCD and was related to descending inhibition/facilitation system and neuroinflammation. Furthermore, behavioral tests, Western blot, and immunofluorescence assay revealed that the pain-relieving effect of Baimai Ointment on CCD may be related to the regulation of the interaction between neuroactive ligand and receptors(neuroligands) such as CHRNA7, ADRA2A, and ADRB2, and the down-regulation of the expression of NOS2/pERK/PI3K, the core regulatory element of HIF-1 signaling pathway in spinal microglia. The findings preliminarily reveal the mechanism of relaxing sinew and activating collaterals of Baimai Ointment in the treatment of Baimai disease, providing a reference for the rational drug use and further research of this agent.
Rats ; Mice ; Animals ; Chronic Pain/metabolism* ; Rats, Sprague-Dawley ; Ganglia, Spinal/metabolism* ; Ligands ; Signal Transduction ; Hyperalgesia/metabolism* ; Drugs, Chinese Herbal

OBJECTIVE: To investigate the inhibitory effect of ANA-12 that blocks brain-derived neurotrophic factor (BDNF)/ tropomyosin receptor kinase B (TrkB) signaling on inflammatory pain in rats and explore the underlying mechanism. METHODS: Forty-two adult SD rats were randomized into BDNF-induced acute pain group (n=24) and CFA-induced chronic pain group. The former group were randomly divided into 4 subgroups, including a control group, ANA-12 treatment group, BDNF treatment group, and BDNF+ANA-12 treatment group; the latter group were subgrouped into control group, CFA treatment group (CFA) and CFA + ANA-12 treatment group. The effects of ANA-12 treatment on pain behaviors of the rats with BDNF-induced acute pain and CFA-induced chronic inflammatory pain were observed. Western blotting was used to examine TrkB signaling and expressions of microglia marker protein Iba1 and TNF-α in the spinal cord of the rats. RESULTS: BDNF injection into the subarachnoid space significantly increased the number of spontaneous paw withdrawal of the rats (P < 0.05), which was obviously reduced by ANA-12 treatment (P < 0.05). The rats with intraplantar injection of CFA, showed significantly increased ipsilateral mechanical stimulation sensitivity (P < 0.05), and ANA-12 treatment obviously increased the ipsilateral foot withdrawal threshold (P < 0.05). Treatment with either BDNF or CFA significantly increased the phosphorylation level of TrkB (Y705) in the spinal cord of the rats (P < 0.05), which was significantly lowered by ANA-12 treatment (P < 0.05). Treatment with BDNF and CFA both significantly up-regulated the expressions of Iba1 and TNF-α in the spinal cord (P < 0.05), but ANA-12 significantly reduced their expression levels (P < 0.05). CONCLUSION ANA-12 can reduce spinal cord inflammation and relieve acute and chronic pain in rats by targeted blocking of BDNF/TrkB signaling.
Animals ; Brain-Derived Neurotrophic Factor/metabolism* ; Chronic Pain/drug therapy* ; Inflammation ; Rats ; Rats, Sprague-Dawley ; Receptor, trkB/metabolism*

The present study was aimed to explore the involvement of dopamine D1 receptor of the anterior cingulate cortex (ACC) in the regulation of chronic inflammatory pain-related emotion. On the first day, the rats were acclimated to the environment and the baseline indices were measured. On the second day, the rats were administered with the dopamine D1 receptor antagonist SCH-23390 or agonist SKF38393 in the ACC, and then they were subcutaneously injected with complete Freund's adjuvant (CFA, 0.08 mL) in the left hind paw to establish conditioned place avoidance (CPA) response after pairing with specific environment. On the third day, the CPA response and the firing frequency of ACC neurons were observed synchronously, and the open-field behavior, mechanical pain behavior and paw withdrawal latency (PWL) tests were also observed subsequently. In other experiments, rats were given subcutaneous injection of normal saline (NS) on the left hind paw after SCH-23390 or SKF-38393 was administered in the ACC, and then the same observations were performed. The results showed that: (1) Compared with the control group, the PWL and mechanical pain thresholds of rats injected with CFA on the left hind paw were significantly decreased (P < 0.05); (2) The residence time of rats injected with CFA in the "pain environment" and open field center was significantly shortened (P < 0.05); (3) Pre-injection of antagonist SCH-23390 in ACC (10 μg) alleviated the anxiety-like negative behavior response induced by CFA (P < 0.05) and reversed CFA-induced increases of discharge frequency of ACC neurons (P < 0.05); (4) Pre-injection of agonist SKF-38393 in the ACC (10 μg) induced CPA-like behavioral response in rats injected with NS in the left hind paw, and increased the firing frequency of ACC neurons (P < 0.05); (5) Immunofluorescence detection showed that dopamine D1 receptor and NMDA receptor were co-expressed in the same neuron. These results suggest that inhibition of dopamine D1 receptor in ACC can alleviate the negative emotional response induced by persistent pain.
2,3,4,5-Tetrahydro-7,8-dihydroxy-1-phenyl-1H-3-benzazepine/adverse effects* ; Animals ; Anxiety ; Chronic Pain ; Gyrus Cinguli ; Hyperalgesia ; Rats ; Receptors, Dopamine D1/metabolism*

We investigated the therapeutic effects of superoxide dismutase (SOD) from thermophilic bacterium HB27 on chronic prostatitis/chronic pelvic pain syndrome (CP/CPPS) and its underlying mechanisms. A Sprague-Dawley rat model of CP/CPPS was prepared and then administered saline or Thermus thermophilic (Tt)-SOD intragastrically for 4 weeks. Prostate inflammation and fibrosis were analyzed by hematoxylin and eosin staining, and Masson staining. Alanine transaminase (ALT), aspartate transaminase (AST), serum creatinine (CR), and blood urea nitrogen (BUN) levels were assayed for all animals. Enzyme-linked immunosorbent assays (ELISA) were performed to analyze serum cytokine concentrations and tissue levels of malondialdehyde, nitric oxide, SOD, catalase, and glutathione peroxidase. Reactive oxygen species levels were detected using dichlorofluorescein diacetate. The messenger ribonucleic acid (mRNA) expression of tissue cytokines was analyzed by reverse transcription polymerase chain reaction (RT-PCR), and infiltrating inflammatory cells were examined using immunohistochemistry. Nuclear factor-κB (NF-κB) P65, P38, and inhibitor of nuclear factor-κBα (I-κBα) protein levels were determined using western blot. Tt-SOD significantly improved histopathological changes in CP/CPPS, reduced inflammatory cell infiltration and fibrosis, increased pain threshold, and reduced the prostate index. Tt-SOD treatment showed no significant effect on ALT, AST, CR, or BUN levels. Furthermore, Tt-SOD reduced inflammatory cytokine expression in prostate tissue and increased antioxidant capacity. This anti-inflammatory activity correlated with decreases in the abundance of cluster of differentiation 3 (CD3), cluster of differentiation 45 (CD45), and macrophage inflammatory protein 1α (MIP1α) cells. Tt-SOD alleviated inflammation and oxidative stress by reducing NF-κB P65 and P38 protein levels and increasing I-κBα protein levels. These findings support Tt-SOD as a potential drug for CP/CPPS.
Animals ; Chronic Pain ; Cytokines/metabolism* ; Fibrosis ; Humans ; Inflammation/metabolism* ; Male ; NF-kappa B/metabolism* ; Pelvic Pain/pathology* ; Prostatitis/metabolism* ; Rats ; Rats, Sprague-Dawley ; Superoxide Dismutase ; Syndrome

Irritable bowel syndrome is a gastrointestinal disorder of unknown etiology characterized by widespread, chronic abdominal pain associated with altered bowel movements. Increasing amounts of evidence indicate that injury and inflammation during the neonatal period have long-term effects on tissue structure and function in the adult that may predispose to gastrointestinal diseases. In this study we aimed to investigate how the epigenetic regulation of DNA demethylation of the p2x7r locus guided by the transcription factor GATA binding protein 1 (GATA1) in spinal astrocytes affects chronic visceral pain in adult rats with neonatal colonic inflammation (NCI). The spinal GATA1 targeting to DNA demethylation of p2x7r locus in these rats was assessed by assessing GATA1 function with luciferase assay, chromatin immunoprecipitation, patch clamp, and interference in vitro and in vivo. In addition, a decoy oligodeoxynucleotide was designed and applied to determine the influence of GATA1 on the DNA methylation of a p2x7r CpG island. We showed that NCI caused the induction of GATA1, Ten-eleven translocation 3 (TET3), and purinergic receptors (P2X7Rs) in astrocytes of the spinal dorsal horn, and demonstrated that inhibiting these molecules markedly increased the pain threshold, inhibited the activation of astrocytes, and decreased the spinal sEPSC frequency. NCI also markedly demethylated the p2x7r locus in a manner dependent on the enhancement of both a GATA1-TET3 physical interaction and GATA1 binding at the p2x7r promoter. Importantly, we showed that demethylation of the p2x7r locus (and the attendant increase in P2X7R expression) was reversed upon knockdown of GATA1 or TET3 expression, and demonstrated that a decoy oligodeoxynucleotide that selectively blocked the GATA1 binding site increased the methylation of a CpG island in the p2x7r promoter. These results demonstrate that chronic visceral pain is mediated synergistically by GATA1 and TET3 via a DNA-demethylation mechanism that controls p2x7r transcription in spinal dorsal horn astrocytes, and provide a potential therapeutic strategy by targeting GATA1 and p2x7r locus binding.
Animals ; Astrocytes/metabolism* ; DNA Demethylation ; Epigenesis, Genetic ; GATA1 Transcription Factor/metabolism* ; Inflammation/metabolism* ; Oligodeoxyribonucleotides/metabolism* ; Rats ; Rats, Sprague-Dawley ; Receptors, Purinergic P2X7/metabolism* ; Visceral Pain/metabolism*

Central sensitization is essential in maintaining chronic pain induced by chronic pancreatitis (CP), but cortical modulation of painful CP remains elusive. Here, we examined the role of the anterior cingulate cortex (ACC) in the pathogenesis of abdominal hyperalgesia in a rat model of CP induced by intraductal administration of trinitrobenzene sulfonic acid (TNBS). TNBS treatment resulted in long-term abdominal hyperalgesia and anxiety in rats. Morphological data indicated that painful CP induced a significant increase in FOS-expressing neurons in the nucleus tractus solitarii (NTS) and ACC, and some FOS-expressing neurons in the NTS projected to the ACC. In addition, a larger portion of ascending fibers from the NTS innervated pyramidal neurons, the neural subpopulation primarily expressing FOS under the condition of painful CP, rather than GABAergic neurons within the ACC. CP rats showed increased expression of vesicular glutamate transporter 1, and increased membrane trafficking and phosphorylation of the N-methyl-D-aspartate receptor (NMDAR) subunit NR2B and the α-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid receptor (AMPAR) subunit GluR1 within the ACC. Microinjection of NMDAR and AMPAR antagonists into the ACC to block excitatory synaptic transmission significantly attenuated abdominal hyperalgesia in CP rats, which was similar to the analgesic effect of endomorphins injected into the ACC. Specifically inhibiting the excitability of ACC pyramidal cells via chemogenetics reduced both hyperalgesia and comorbid anxiety, whereas activating these neurons via optogenetics failed to aggravate hyperalgesia and anxiety in CP rats. Taken together, these findings provide neurocircuit, biochemical, and behavioral evidence for involvement of the ACC in hyperalgesia and anxiety in CP rats, as well as novel insights into the cortical modulation of painful CP, and highlights the ACC as a potential target for neuromodulatory interventions in the treatment of painful CP.
Animals ; Anxiety/etiology* ; Chronic Pain/etiology* ; GABAergic Neurons ; Gyrus Cinguli/metabolism* ; Hyperalgesia/metabolism* ; Pancreatitis, Chronic/pathology* ; Rats ; Rats, Sprague-Dawley ; Receptors, N-Methyl-D-Aspartate/metabolism* ; Trinitrobenzenesulfonic Acid/toxicity*