Rat Spinal Dorsal Horn Research Articles

Ferroptosis Regulated by 5-HT3a Receptor via Calcium/Calmodulin Signaling Contributes to Neuropathic Pain in Brachial Plexus Avulsion Rat Models.

Neuropathic pain is a prevalent complication following brachial plexus avulsion (BPA). Ferroptosis has been implicated in various nervous system disorders. However, the association between ferroptosis and neuropathic pain induced by BPA remains unclear. This study aimed to investigate the role of ferroptosis in BPA-induced neuropathic pain. A rat model of neuropathic pain was established via BPA induction. Pain thresholds of rats were measured after BPA surgery and intraperitoneal injection of Fer-1. On day 14 postsurgery, spinal dorsal horn (SDH) samples were collected for Western blotting, biochemical analysis, and immunohistochemistry to analyze the expression and distribution of ferroptosis-related markers. The relationships among 5-HT3a receptor, calcium/calmodulin (CaM) pathway, and ferroptosis were assessed via Western blotting, biochemical analysis, and lipid peroxidation assays, including iron and calcium content, reactive oxygen species, glutathione peroxidase 4 (GPX4), ACSL, and CaM expression. BPA-induced neuropathic pain was associated with iron accumulation, increased lipid peroxidation, dysregulated expression of Acyl-CoA synthetase long-chain family member 4, and GPX4, and changes in transferrin receptor, divalent metal transporter 1, and ferroportin-1 (FPN1). Intraperitoneal administration of Fer-1 reversed all of these alterations and mitigated mechanical and cold hypersensitivity. Inhibition of the 5-HT3a receptor reduced the extent of ferroptosis. Furthermore, the 5-HT3a receptor can regulate the calcium/CaM pathway via L-type calcium channels (LTCCs), and blocking LTCCs with nifedipine also alleviated ferroptosis in the SDH of BPA rats. Taken together, in rats with BPA, the development of neuropathic pain involves ferroptosis, which is regulated by the 5-HT3a receptor through the LTCCs and the calcium/CaM signaling pathway in the SDH.

(+)-Catechin attenuates CCI-induced neuropathic pain in male rats by promoting the Nrf2 antioxidant pathway to inhibit ROS/TLR4/NF-κB-mediated activation of the NLRP3 inflammasome.

The objective of this study was to investigate the potential mechanisms by which (+)-catechin alleviates neuropathic pain. Thirty-two male Sprague-Dawley rats were divided into four groups: the sham group, the chronic constriction injury (CCI)group, the CCI+ ibuprofen group, and the CCI+ (+)-catechin group. CCI surgery induces thermal hyperalgesia in rats and (+)-catechin ameliorated CCI-induced thermal hyperalgesia and repaired damaged sciatic nerve in rats. CCI decreased SOD levels in male rat spinal cord dorsal horn and promoted MDA production, induced oxidative stress by increasing NOX4 levels and decreasing antioxidant enzyme HO-1 levels, and also increased protein levels of TLR4, p-NF-κB, NLRP3 inflammasome components, and IL-1β. In contrast, (+)-catechin reversed the above results. In i vitro experiments, (+)-catechin reduced the generation of reactive oxygen species (ROS) in GMI-R1 cells after LPS stimulation and attenuated the co-expression of IBA-1 and NLRP3. It also showed significant inhibition of the NF-κB and NLRP3 inflammatory pathways and activation of the Nrf2-mediated antioxidant system. Overall, these findings suggest that (+)-catechin inhibits the activation of the NLRP3 inflammasome through the triggering of the Nrf2-induced antioxidant system, the inhibition of the TLR4/NF-κB pathway, and the production of ROS to alleviate CCI-induced neuropathic pain in male rats.

Photobiomodulation transiently increases the spontaneous firing in the superficial layer of the rat spinal dorsal horn

The therapeutic benefits of photobiomodulation (PBM) in pain management, although well documented, are accompanied by concerns about potential risks, including pain, particularly at higher laser intensities. This study investigated the effects of laser intensity on pain perception using behavioral and electrophysiological evaluations in rats. Our results show that direct laser irradiation of 1000 mW/cm2 to the sciatic nerve transiently increases the frequency of spontaneous firing in the superficial layer without affecting the deep layer of the spinal dorsal horn, and this effect reverses to pre-irradiation levels after irradiation. Interestingly, laser irradiation at 1000 mW/cm2, which led to an increase in spontaneous firing, did not prompt escape behavior. Furthermore, a significant reduction in the time to initiate escape behavior was observed only at 9500 mW/cm2 compared to 15, 510, 1000, and 4300 mW/cm2. This suggests that 1000 mW/cm2, the laser intensity at which an increase in spontaneous firing was observed, corresponds to a stimulus that did not cause pain. It is expected that a detailed understanding of the risks and mechanisms of PBM from a neurophysiological perspective will lead to safer and more effective use of PBM.

Actions of remimazolam on inhibitory transmission of rat spinal dorsal horn neurons

Remimazolam is an ultra-short benzodiazepine that acts on the benzodiazepine site of γ-aminobutyric acid (GABA) receptors in the brain and induces sedation. Although GABA receptors are found localized in the spinal dorsal horn, no previous studies have reported the analgesic effects or investigated the cellular mechanisms of remimazolam on the spinal dorsal horn. Behavioral measures, immunohistochemistry, and in vitro whole-cell patch-clamp recordings of dorsal horn neurons were used to assess synaptic transmission. Intrathecal injection of remimazolam induced behavioral analgesia in inflammatory pain-induced mechanical allodynia (six rats/dose; p < 0.05). Immunohistochemical staining revealed that remimazolam suppressed spinal phosphorylated extracellular signal-regulated kinase activation (five rats/group, p < 0.05). In vitro whole-cell patch-clamp analysis demonstrated that remimazolam increased the frequency of GABAergic miniature inhibitory post-synaptic currents, prolonged the decay time (six rats; p < 0.05), and enhanced GABA currents induced by exogenous GABA (seven rats; p < 0.01). However, remimazolam did not affect miniature excitatory post-synaptic currents or amplitude of monosynaptic excitatory post-synaptic currents evoked by Aδ- and C-fiber stimulation (seven rats; p > 0.05). This study suggests that remimazolam induces analgesia by enhancing GABAergic inhibitory transmission in the spinal dorsal horn, suggesting its potential utility as a spinal analgesic for inflammatory pain.

Photobiomodulation inhibits neuronal firing in the superficial but not deep layer of a rat spinal dorsal horn

Photobiomodulation (PBM) has attracted attention as a treatment for chronic pain. Previous studies have reported that PBM of the sciatic nerve inhibits neuronal firing in the superficial layers (lamina I–II) of the spinal dorsal horn of rats, which is evoked by mechanical stimulation that corresponds to noxious stimuli. However, the effects of PBM on the deep layers (lamina III–IV) of the spinal dorsal horn, which receive inputs from innocuous stimuli, remain poorly understood. In this study, we examined the effect of PBM of the sciatic nerve on firing in the deep layers of the spinal dorsal horn evoked by mechanical stimulation. Before and after PBM, mechanical stimulation was administered to the cutaneous receptive field using 0.6–26.0 g von Frey filaments (vFFs), and vFF-evoked firing in the deep layers of the spinal dorsal horn was recorded. The vFF-evoked firing frequencies were not altered after the PBM for any of the vFFs. The inhibition rate for 26.0 g vFF-evoked firing was approximately 13 % in the deep layers and 70 % in the superficial layers. This suggests that PBM selectively inhibits the transmission of pain information without affecting the sense of touch. PBM has the potential to alleviate pain while preserving the sense of touch.

Sirtuin 3 Mediated by Spinal cMyc-Enhancer of Zeste Homology 2 Pathway Plays an Important Role in Human Immunodeficiency Virus-Related Neuropathic Pain Model.

Clinical data demonstrate that chronic use of opioid analgesics increases neuropathic pain in people living with human immunodeficiency virus (HIV). Therefore, it is important to elucidate the molecular mechanisms of HIV-related chronic pain. In this study, we investigated the role of the transcription factor cMyc, epigenetic writer enhancer of zeste homology 2 (EZH2), and sirtuin 3 (Sirt3) pathway in HIV glycoprotein gp120 with morphine (gp120M)-induced neuropathic pain in rats. Neuropathic pain was induced by intrathecal administration of recombinant gp120 with morphine. Mechanical withdrawal threshold was measured using von Frey filaments, and thermal latency using the hotplate test. Spinal expression of cMyc, EZH2, and Sirt3 were measured using Western blots. Antinociceptive effects of intrathecal administration of antisense oligodeoxynucleotide against cMyc, a selective inhibitor of EZH2, or recombinant Sirt3 were tested. In the spinal dorsal horn, gp120M upregulated expression of cMyc (ratio of gp120M versus control, 1.68 ± 0.08 vs 1.00 ± 0.14, P = .0132) and EZH2 (ratio of gp120M versus control, 1.76 ± 0.05 vs 1.00 ± 0.16, P = .006), and downregulated Sirt3 (ratio of control versus gp120M, 1.00 ± 0.13 vs 0.43 ± 0.10, P = .0069) compared to control. Treatment with intrathecal antisense oligodeoxynucleotide against cMyc, GSK126 (EZH2 selective inhibitor), or recombinant Sirt3 reduced mechanical allodynia and thermal hyperalgesia in this gp120M pain model. Knockdown of cMyc reduced spinal EZH2 expression in gp120M treated rats. Chromatin immunoprecipitation (ChIP) assay showed that enrichment of cMyc binding to the ezh2 gene promoter region was increased in the gp120M-treated rat spinal dorsal horn, and that intrathecal administration of antisense ODN against cMyc (AS-cMyc) reversed the increased enrichment of cMyc. Enrichment of trimethylation of histone 3 on lysine residue 27 (H3K27me3; an epigenetic mark associated with the downregulation of gene expression) binding to the sirt3 gene promoter region was upregulated in the gp120M-treated rat spinal dorsal horn; that intrathecal GSK126 reversed the increased enrichment of H3K27me3 in the sirt3 gene promoter. Luciferase reporter assay demonstrated that cMyc mediated ezh2 gene transcription at the ezh2 gene promoter region, and that H3K27me3 silenced sirt3 gene transcription at the gene promoter region. These results demonstrated that spinal Sirt3 decrease in gp120M-induced neuropathic pain was mediated by cMyc-EZH2/H3K27me3 activity in an epigenetic manner. This study provided new insight into the mechanisms of neuropathic pain in HIV patients with chronic opioids.

5-HT3a receptor contributes to neuropathic pain by regulating central sensitization in a rat with brachial plexus avulsion

PurposeAs a frequently occurring complication resulting from brachial plexus avulsion (BPA), neuropathic pain significantly impacts the quality of life of patients and places a substantial burden on their families. Recent reports have suggested that the 5-HT3a receptor may play a role in the development and regulation of neuropathic pain. The current study aimed to explore the involvement of the 5-HT3a receptor in neuropathic pain resulting from BPA in rats. MethodsA rat model of neuropathic pain was induced through brachial plexus avulsion (BPA). The pain thresholds of the rats were measured after BPA. The spinal dorsal horn (SDH) of rats was collected at day 14 after surgery, and the expression and distribution of the 5-HT3a receptor were analyzed using immunohistochemistry and western blotting. The expression levels of various factors related to central sensitization were measured by western blot, including c-Fos, GFAP, IBA-1, IL-1β and TNF-α. The effects of 5-HT3a receptor antagonists on hyperalgesia were assessed through behavioral tests after intrathecal administration of ondansetron. Additionally, at 120 min postinjection, the SDH of rats was acquired, and the change of expression levels of protiens related to central sensitization were measured by western blot. ResultsBPA induced mechanical and cold hypersensitivity in rats. The 5-HT3a receptor was increased and mainly distributed on neurons and microglia in the SDH after BPA, and the level of central sensitization and expression of inflammatory factors, such as c-Fos, GFAP, IBA-1, IL-1β and TNF-α, were also increased markedly. Ondansetron, which is a selective 5-HT3a receptor antagonist, reversed the behavioral changes caused by BPA. The antagonist also decreased the expression of central sensitization markers and inflammatory factors. ConclusionThe results suggested that the 5-HT3a receptor is involved in neuropathic pain by regulating central nervous system sensitization in a rat brachial plexus avulsion model. Targeting the 5-HT3a receptor may be a promising approach for treating neuropathic pain after brachial plexus avulsion.

Massage ameliorates lumbar disc herniation-related radicular pain in rats by suppressing TLR4/NLRP3 inflammasome signaling transduction.

Lumbar disc herniation (LDH) is a common spinal disease that can cause severe radicular pain. Massage, also known as Tuina in Chinese, has been indicated to exert an analgesic effect in patients with LDH. Nonetheless, the mechanism underlying this effect of massage on LDH remains unclarified. Forty Sprague-Dawley rats were randomly divided into four groups. A rat LDH model was established by autologous nucleus pulpous (NP) implantation, followed by treatment with or without massage. A toll-like receptor 4 (TLR4) antagonist TAK-242 was administrated to rats for blocking TLR4. Behavioral tests were conducted to examine rat mechanical and thermal sensitivities. Western blotting was employed for determining TLR4 and NLRP3 inflammasome-associated protein levels in the spinal dorsal horn (SDH). Immunofluorescence staining was implemented for estimating the microglial marker Iba-1 expression in rat SDH tissue. NP implantation induced mechanical allodynia and thermal hyperalgesia in rat ipsilateral hindpaws and activated TLR4/NLRP3 inflammasome signaling transduction in the ipsilateral SDH. Massage therapy or TAK-242 administration relieved NP implantation-triggered pain behaviors in rats. Massage or TAK-242 hindered microglia activation and blocked TLR4/NLRP3 inflammasome activation in ipsilateral SDH of LDH rats. Massage ameliorates LDH-related radicular pain in rats by suppressing microglia activation and TLR4/NLRP3 inflammasome signaling transduction.

Methylglyoxal activates transient receptor potential A1/V1 via reactive oxygen species in the spinal dorsal horn.

Methylglyoxal (MGO), a highly reactive dicarbonyl metabolite of glucose primarily formed during the glycolytic pathway, is a precursor of advanced glycation end-products (AGEs). Recently, numerous studies have shown that MGO accumulation can cause pain and hyperalgesia. However, the mechanism through which MGO induces pain in the spinal dorsal horn remains unclear. The present study investigated the effect of MGO on spontaneous excitatory postsynaptic currents (sEPSC) in rat spinal dorsal horn neurons using blind whole-cell patch-clamp recording. Perfusion of MGO increased the frequency and amplitude of sEPSC in spinal horn neurons in a concentration-dependent manner. Additionally, MGO administration increased the number of miniature EPSC (mEPSC) in the presence of tetrodotoxin, a sodium channel blocker. However, 6-cyano-7-nitroqiunocaline-2,3-dione (CNQX), an AMPA/kainate receptor antagonist, blocked the enhancement of sEPSC by MGO. HC-030031, a TRP ankyrin-1 (TRPA1) antagonist, and capsazepine, a TRP vanilloid-1 (TRPV1) antagonist, inhibited the action of MGO. Notably, the effects of MGO were completely inhibited by HC-030031 and capsazepine. MGO generates reactive oxygen species (ROS) via AGEs. ROS also potentially induce pain via TRPA1 and TRPV1 in the spinal dorsal horn. Furthermore, we examined the effect of MGO in the presence of N-tert-butyl-α-phenylnitrone (PBN), a non-selective ROS scavenger, and found that the effect of MGO was completely inhibited. These results suggest that MGO increases spontaneous glutamate release from the presynaptic terminal to spinal dorsal horn neurons through TRPA1, TRPV1, and ROS and could enhance excitatory synaptic transmission.

Effect of Reboxetine Treatment on BDNF, Synaptophysin, and PSD-95 Levels in the Spinal Dorsal Horn of Rats with Diabetic Neuropathy

Objective: It is known that neuropathic pain is accompanied by alterations in the levels of neurotrophic factors and synaptic proteins in the microenvironment of the spinal dorsal horn. Such changes contribute to hyperalgesia and allodynia processes; thus, analgesic drugs can exert their pharmacological effects by affecting the expressions, levels, or functions of these endogenous substances. In this study, based on the knowledge that reboxetine (a selective noradrenaline reuptake inhibitor) has the potential for antihyperalgesic efficacy in diabetic neuropathy, we aimed to examine the probable effects of this drug on diabetes-induced changes in brain-derived neurotrophic factor (BDNF), synaptophysin (the pre-synaptic marker of synaptic integration), and postsynaptic density-95 (PSD-95) (the postsynaptic marker of synaptic integration) levels in the superficial laminae of the dorsal horn. &#x0D; Methods: Experimental diabetes was induced by a single-dose injection of streptozotocin (STZ) (50 mg/kg) in rats. After four week-long induction period of painful diabetic neuropathy, rats were treated orally with 8 mg/kg reboxetine for two weeks. Hyperalgesia responses were evaluated by using the Randall–Selitto and Hargreave's tests. Following the pain tests, immunohistochemical studies were performed.&#x0D; Results: Two weeks of reboxetine administration increased the reduced paw withdrawal thresholds and shortened the paw withdrawal latencies of diabetic rats in neuropathic pain tests, indicating the antihyperalgesic efficacy of this drug. Moreover, augmented BDNF and synaptophysin levels in diabetic rats reversed by reboxetine treatment. However, there was no alteration in the densities of PSD-95, in both STZ-diabetic and reboxetine-treated STZ-diabetic rats. &#x0D; Conclusion: The obtained results suggested that inhibition of central sensitization and modulation of spinal plasticity seem to be pharmacological mechanisms underlying reboxetine's antihyperalgesic effects on diabetic rats. However, further studies are still needed to clarify the exact mechanism of action.

Spinal TAOK2 contributes to neuropathic pain via cGAS-STING activation in rats

Thousand and one amino acid kinase 2 (TAOK2) is a member of the mammalian sterile 20 kinase family and is implicated in neurodevelopmental disorders; however, its role in neuropathic pain remains unknown. Here, we found that TAOK2 was enriched and activated after chronic constriction injury (CCI) in the rat spinal dorsal horn. Meanwhile, cyclic guanosine monophosphate-adenosine monophosphate synthase (cGAS)-stimulator of interferon genes (STING) signaling was also activated with hyperalgesia. Silencing TAOK2 reversed hyperalgesia and suppressed the activation of cGAS-STING signaling induced by CCI, while pharmacological activation of TAOK2 induced pain hypersensitivity and upregulation of cGAS-STING signaling in naive rats. Furthermore, pharmacological inhibition or gene silencing of cGAS-STING signaling attenuated CCI-induced hyperalgesia. Taken together, these data demonstrate that the activation of spinal TAOK2 contributes to CCI-induced hyperalgesia via cGAS-STING signaling activation, providing new molecular targets for the treatment of neuropathic pain.

Relationship between Laser Intensity at the Peripheral Nerve and Inhibitory Effect of Percutaneous Photobiomodulation on Neuronal Firing in a Rat Spinal Dorsal Horn.

Photobiomodulation is an effective treatment for pain. We previously reported that the direct laser irradiation of the exposed sciatic nerve inhibited firing in the rat spinal dorsal horn evoked by mechanical stimulation, corresponding to the noxious stimulus. However, percutaneous laser irradiation is used in clinical practice, and it is unclear whether it can inhibit the firing of the dorsal horn. In this study, we investigated whether the percutaneous laser irradiation of the sciatic nerve inhibits firing. Electrodes were inserted into the lamina II of the dorsal horn, and mechanical stimulation was applied using von Frey filaments (vFFs) with both pre and post laser irradiation. Our findings show that percutaneous laser irradiation inhibited 26.0 g vFF-evoked firing, which corresponded to the noxious stimulus, but did not inhibit 0.6 g and 8.0 g vFF-evoked firing. The post- (15 min after) and pre-irradiation firing ratios were almost the same as those for direct and percutaneous irradiation. A photodiode sensor implanted in the sciatic nerve showed that the power density reaching the sciatic nerve percutaneously was attenuated to approximately 10% of that on the skin. The relationship between the laser intensity reaching the nerve and its effect could be potentially useful for a more appropriate setting of laser conditions in clinical practice.

Electroacupuncture relieves chronic pain by promoting microglia M2 polarization in lumbar disc herniation rats.

Electroacupuncture has an effective analgesia on chronic pain caused by lumbar disc herniation (LDH) clinically, however, the underlying mechanism is unclear. In this study, we investigated whether electroacupuncture alleviated pain in LDH model rats by inducing spinal microglia M2 polarization. We established a noncompression LDH rat model by implanting autologous caudal nucleus pulposus into L5/L6 nerve root. Electroacupuncture (30 min/day) treatment on the ipsilateral side was started on the 8th postoperative day, once a day for consecutive 7 days. Paw withdrawal threshold (PWT) and paw withdrawal latency (PWL) were tested for pain behavior. Western blotting was used to detect the protein expression in lumbar enlargement (L5/L6). Immunofluorescence was used to detect iNOS+/Iba-1+ and Arg-1+/Iba-1+ and CB2R+/Iba-1+ in lumbar enlargement (L5/L6). We show that PWT and PWL decreased in the LDH group while Iba-1, iNOS, and TNF-α expression increased significantly in lumbar spinal dorsal horn (SDH) after LDH surgery, and revealing that microglia were activated and polarized towards proinflammatory M1 phenotype. Electroacupuncture treatment significantly increased PWT and PWL while reducing Iba-1, iNOS, and TNF-α expression, interestingly, Arg-1 and IL-10 expression were significantly increased. Moreover, electroacupuncture treatment led to CB2 receptors on microglia upregulation, while NF-κB and p-NF-κB expression in lumbar SDH downregulation. Our study indicated that electroacupuncture may reduce nociceptive hyperalgesia by inhibiting microglia activation and microglia M1 polarization and promoting microglia M2 polarization in lumbar SDH of LDH rats, which may be caused by the activation of CB2 receptors on microglia and inhibition of NF-κB pathway in lumbar SDH.

Intervention effect of Tuina pressing and kneading the Huantiao (GB30) acupoint on NF-κB p65 protein at spinal cord dorsal horn in sciatica rats

To observe the analgesic effect of Tuina by pressing and kneading the Huantiao (GB30) acupoint on rats with chronic constriction injury (CCI) and to explore the analgesic mechanism of Tuina on sciatica rats. Thirty-two SPF male SD rats weighing 180 to 220 g were randomly divided into fore groups:blank group (without any treatment), sham group (only exposed without sciatic nerve ligating), model group (sciatic nerve ligating) and Tuina group (manual intervention after lsciatic nerve ligating). The CCI model was prepared by ligating the right sciatic nerve of the rats, on the third day of modeling, the rats in the Tuina group were given pressing and kneading the Huantiao (GB30) point for 14 days, and the changes of paw withdrawal threshold(PWT), paw withdrawal latency(PWL) were measured before and on the 1st, 3rd, 7th, 10th, 14th and 17th days after modeling. The changes of sciatic functional index(SFI) were measured before and on the 1st and 17th day after modeling. The morphological changes of the sciatic nerve were observed by hematoxylin-eosin(HE) staining;and the differences in NF-κB protein expression in the right dorsal horn of the spinal cord of rats were detected. Following modeling, there was no significant difference in PWT, PWL and SFI between the blank group and the sham group (P>0.05), but the PWT, PWL and SFI of the model group and the Tuina group decreased significantly (P<0.01). After manual intervention, the pain threshold of rats in Tuina group increased. On the 8th day of manual intervention (the 10th day after modeling), PWT in Tuina group increased significantly compared with that in model group (P<0.01). On the 5th day of manual intervention (the 7th day after modeling), the PWL of the massage group was significantly higher than that of the model group (P<0.01). The pain threshold of rats in Tuina group continued to rise with the continuous manipulation intervention. After 14 days of manipulative intervention, the sciatic nerve function index of rats in the Tuina group increased significantly(P<0.01). Compared with the blank group and sham group, the myelinated nerve fibers of sciatic nerve in the model group were disordered and the density of axons and myelin sheath was uneven. Compared with the model group, the nerve fibers of rats in the Tuina group were gradually continuous and the axons and myelin sheath were more uniform than those in the model group. Compared with the blank group and sham group, the expression of NF-κB protein in the right spinal dorsal horn of the model group was significantly increased(P<0.01). Compared with the model group, the expression of NF-κB protein in the right spinal dorsal horn of rats in Tuina group decreased significantly(P<0.01). Pressing and kneading the Huantiao (GB30) point restores nerve fiber alignment;and improves the PWT、PWL and SFI in the CCI model by decreasing NF-κB p65 protein expression in the spinal dorsal horn. There fore, Tuina demmstrates an analgesic effect and improves the gait of rats with sciatica.

Upregulation of lysine-specific demethylase 6B aggravatesinflammatory pain through H3K27me3 demethylation-dependent production of TNF-α in the dorsal root ganglia and spinal dorsal horn in rats.

Lysine-specific demethylase 6B (KDM6B) serves as a key mediator of gene transcription. It regulates expression of proinflammatory cytokines and chemokines in variety of diseases. Herein, the role and the underlying mechanisms of KDM6B in inflammatory pain were studied. The inflammatory pain was conducted by intraplantar injection of complete Freund's adjuvant (CFA) in rats. Immunofluorescence, Western blotting, qRT-PCR, and chromatin immunoprecipitation (ChIP)-PCR were performed to investigate the underlying mechanisms. CFA injection led to upregulation of KDM6B and decrease in the level of H3K27me3 in the dorsal root ganglia (DRG) and spinal dorsal horn. The mechanical allodynia and thermal hyperalgesia following CFA were alleviated by the treatment of intrathecal injection of GSK-J4, and by microinjection of AAV-EGFP-KDM6B shRNA in the sciatic nerve or in lumbar 5 dorsal horn. The increased production of tumor necrosis factor-α (TNF-α) following CFA in the DRGs and dorsal horn was inhibited by these treatments. ChIP-PCR showed that CFA-induced increased binding of nuclear factor κB with TNF-α promoter was repressed by the treatment of microinjection of AAV-EGFP-KDM6B shRNA. These results suggest that upregulated KDM6B via facilitating TNF-α expression in the DRG and spinal dorsal horn aggravates inflammatory pain.

Epitranscriptomic profiling of N4-acetylcytidine-related RNA acetylation in the spinal dorsal horn of rat with cancer-induced bone pain.

Recently, epigenetics involved in the regulation of gene expression has become a research hotspot. This study evaluated N4-acetylcytidine (ac4c) RNA acetylation in the spinal dorsal horn (SDH) of rats with cancer-induced bone pain (CIBP). The ac4C-specific RIP sequencing and NAT10-specific RIP sequencing were performed to identify the differences in ac4C acetylation and gene expression in the SDH between CIBP and sham groups, the relationship with the acetylation-modifying enzyme NAT10, and association analysis was performed. By interfering with the NAT10 expression, the relationship between some up-regulated genes and ac4C acetylation in CIBP was verified. In this study, we demonstrated that bone cancer increases the levels of NAT10 and the overall acetylation, inducing differential ac4C patterns in the SDH of rats. Through verification experiments, it was found that ac4C acetylation of some genes is regulated by NAT10, and differential ac4C patterns in RNA determine the expression of this RNA. We exposed that some CIBP-related gene expression was altered in the SDH of rats, which was regulated by differentially expressed ac4C acetylation.

Folic acid relieves bone cancer pain by downregulating P2X2/3 receptors in rats

BackgroundBone cancer pain (BCP) remains a clinical challenge due to the limited and side effects of therapeutic methods. Folic acid has been known as an FDA approved dietary supplement and proved to have an analgesic effect in neuropathic pain. Here we investigate the role and mechanism of folic acid in bone cancer pain of a rat model. MethodsWalker 256 tumor cells were inoculated into the left tibia of rats to induce bone cancer pain model. Pain reflex were assessed by paw withdrawal threshold (PWT) response to Von Frey filaments and paw withdrawal latency (PWL) response to thermal stimulation. Folic acid was injected intraperitoneally to evaluate its analgesic effect in rats with bone cancer pain. Western blotting and qPCR were used to determine P2X2/3 receptor protein and mRNA levels in ipsilateral L4-6 dorsal root ganglion (DRG) and spinal dorsal horn (SDH). ResultsThe PWT and PWL of rats with bone cancer pain were obviously decreased compared to the naïve and sham rats. Interestingly, continuous folic acid treatment significantly increased the PWT and PWL of rats with bone cancer pain. P2X2 and P2X3 receptors were clearly upregulated at both mRNA and protein expression in L4-6 DRG and SDH of rats with bone cancer pain. P2X2 and P2X3 receptors were mainly localized with CGRP (calcitonin gene-related peptide) or IB4 (isolectin B4) positive neurons in L4-6 DRG of rats with bone cancer pain. Notably, continuous folic acid treatment significantly reduced the expression of P2X2 and P2X3 receptors in L4-6 DRG and SDH of rats with bone cancer pain. Finally, intrathecal injection of A317491 (a selective antagonist of P2X2/3 receptors) markedly elevated the PWT and PWL of rats with bone cancer pain. ConclusionThese results suggest that folic acid has an effective antinociceptive effect on bone cancer pain, which is mediated by downregulating P2X2/3 receptors in L4-6 DRG and SDH of rats with bone cancer pain. Folic acid may be a novel therapeutic strategy in cancer patients for pain relief.

Exploring the effect of Tuina on the dendritic structure of spinal cord dorsal horn in rats with lumbar disc herniation based on NR2B/PSD-95 pathway

ObjectiveTo investigate the analgesic mechanism of Tuina (Chinese therapeutic massage) by observing the effect of the N-methyl-D-aspartate receptor subunit 2B (NR2B)/postsynaptic density-95 (PSD-95) pathway on the dendritic structure of spinal cord dorsal horn in rats with lumbar disc herniation.MethodsFifty Sprague-Dawley rats were randomly divided into a blank group, a model group, a Tuina group, a blocker agent group, and a blocker agent + Tuina group. The sciatic nerve chronic constriction injury (CCI) model was prepared by the sciatic nerve ligation method. From the 4th day after modeling, rats in the Tuina group and the blocker agent + Tuina group were subject to daily Tuina intervention, and those in the blocker agent group and the blocker agent + Tuina group were daily intrathecally injected with NR2B blocker agent (MK-801). The spontaneous pain score was used to observe the pain behavior of all rats. The expression levels of NR2B and downstream PSD-95 were measured by immunohistochemistry, and the dendritic structure changes were observed by Golgi staining for rat spinal cord dorsal horn after 14 d of continuous intervention.ResultsCompared with the blank group, the degree of rat spontaneous pain after CCI was elevated in both the model and the Tuina groups (P<0.01) and was reduced in the Tuina group after the Tuina intervention compared with the model group (P<0.05). Compared with the model group, the rat spontaneous pain level after blocking NR2B was reduced in both the blocker agent group and the blocker agent + Tuina group (P<0.05). The NR2B and PSD-95 protein levels were significantly higher in the model group compared with the blank group (P<0.01); the total number of dendritic branches was increased (P<0.01), and the total dendritic length became longer (P<0.01) in the spinal cord dorsal horn. The rat NR2B and PSD-95 protein levels were significantly decreased in the Tuina group compared with the model group (P<0.01); the total dendritic branch number was reduced (P<0.01) and the total length was shortened (P<0.01) in the spinal cord dorsal horn. After blocking NR2B, the expression levels of NR2B and downstream PSD-95 protein were significantly lower in both the blocker agent group and the blocker agent + Tuina group compared to the model group (P<0.01). The total branch number was significantly reduced (P<0.01), and the total length was significantly shortened (P<0.01) of the dendrites in the spinal cord dorsal horn.ConclusionTuina may exert an analgesic effect by remodeling the dendritic structure in the spinal cord dorsal horn in rats with lumbar disc herniation, and its mechanism may be related to the inhibition of NR2B/PSD-95 signaling pathway.

Neuronal P2X4 receptor may contribute to peripheral inflammatory pain in rat spinal dorsal horn.

Intense inflammation may result in pain, which manifests as spinal central sensitization. There is growing evidence that purinergic signaling plays a pivotal role in the orchestration of pain processing. Over the last decade the ionotropic P2X purino receptor 4 (P2X4) got into spotlight in neuropathic disorders, however its precise spinal expression was scantily characterized during inflammatory pain. Thus, we intended to analyze the receptor distribution within spinal dorsal horn and lumbar dorsal root ganglia (DRG) of rats suffering in inflammatory pain induced by complete Freund adjuvant (CFA). CFA-induced peripheral inflammation was validated by mechanical and thermal behavioral tests. In order to ensure about the putative alteration of spinal P2X4 receptor gene expression qPCR reactions were designed, followed by immunoperoxidase and Western blot experiments to assess changes at a protein level. Colocalization of P2X4 with neuronal and glial markers was investigated by double immunofluorescent labelings, which were subsequently analyzed with IMARIS software. Transmission electronmicroscopy was applied to study the ultrastructural localization of the receptor. Concurrently, in lumbar DRG cells similar methodology has been carried out to complete our observations. The figures of mechanical and thermal behavioral tests proved the establishment of CFA-induced inflammatory pain. We observed significant enhancement of P2X4 transcript level within the spinal dorsal horn 3 days upon CFA administration. Elevation of P2X4 immunoreactivity within Rexed lamina I-II of the spinal gray matter was synchronous with mRNA expression, and confirmed by protein blotting. According to IMARIS analysis the robust protein increase was mainly detected on primary afferent axonterminals and GFAP-labelled astrocyte membrane compartments, but not on postsynaptic dendrites was also validated ultrastructurally within the spinal dorsal horn. Furthermore, lumbar DRG analysis demonstrated that peptidergic and non-peptidergic nociceptive subsets of ganglia cells were also abundantly positive for P2X4 receptor in CFA model. Here we provide novel evidence about involvement of neuronal and glial P2X4 receptor in the establishment of inflammatory pain.

Spinal cannabinoid receptor 2 activation alleviates neuropathic pain by regulating microglia and suppressing P2X7 receptor.

Neuropathic pain (NP) is the chronic pain in patients resulting from injuries or diseases in the somatosensory nervous system. However, effective treatment remains limited to opioids. Currently, there is an urgent need to develop new specific pharmaceuticals with low abuse potentiality. Cannabinoid receptor 2 (CB2R) is one of the significant receptors in the endocannabinoid system. It is widely expressed in the central nervous system, especially enriched in glial cells, and plays an important role in the occurrence and development of inflammation in the nervous system. CB2R activation has a neuroprotective effect on nerve injury. In this study, we report increased and more reactive microglia (with larger cell body, shorter processes, and fewer endpoints) observed in the spinal dorsal horn of spared nerve injury (SNI) rats. Continuous intrathecal administration of CB2R agonist PM226 attenuated mechanical and cold hyperalgesia in rats and prevented the transition of microglia to the proinflammatory stage. Thus, microglia transitioned into the neuroprotective stage. Meanwhile, the proinflammatory factors TNF-α and iNOS decreased, and the levels of anti-inflammatory factors Arg-1 and IL-10 increased. The content of P2X7 receptors in the spinal dorsal horn of rats increases with time after SNI. After continuous intrathecal administration of PM226, the content of P2X7 protein decreases significantly. The administration of P2X7 inhibitor A-438079 alleviated the mechanical hyperalgesia of rats, reduced the number of microglia, and decreased the content of P2X7. These results indicate that P2X7 is involved in the neuroprotective effect caused by CB2R activation. In conclusion, this study provides new insights into the neuroprotective mechanism of CB2R activation.