Dorsal Horn Neurons Research Articles

Interaction between dorsal horn neuron subsets operated by a neuropeptide Y and prodynorphin promoter and its contribution to Aβ fiber–induced allodynia–like behavior in rats

Interaction between dorsal horn neuron subsets operated by a neuropeptide Y and prodynorphin promoter and its contribution to Aβ fiber–induced allodynia–like behavior in rats

Euchromatin histone-lysine N-methyltransferase 2 regulates the expression of potassium-sodium-activated channel subfamily T member 1 in primary sensory neurons and contributes to remifentanil-induced pain sensitivity

Intraoperative remifentanil administration has been linked to increased postoperative pain sensitivity. Recent studies have identified the involvement of euchromatic histone-lysine N-methyltransferase 2 (Ehmt2/G9a) in neuropathic pain associated with the transcriptional silencing of many potassium ion channel genes. This study investigates whether G9a regulates the potassium sodium-activated channel subfamily T member 1 (Slo2.2) in remifentanil-induced post-incisional hyperalgesia (RIH) in rodents. We performed remifentanil infusion (1 μg·kg-1·min-1 for 60 min) followed by plantar incision to induce RIH in rodents. Our results showed that RIH was accompanied by increased G9a and H3K9me2 production and decreased Slo2.2 expression 48 h postoperatively. Deletion of G9a rescued Slo2.2 expression in DRG and reduced RIH intensity. Slo2.2 overexpression also reversed this hyperalgesia phenotype. G9a overexpression decreased Slo2.2-mediated leak current and increased excitability in the small-diameter DRG neurons and laminal II small-diameter neurons in the spinal dorsal horn, which was implicated in peripheral and central sensitization. These results suggest that G9a contributes to the development of RIH by epigenetically silencing Slo2.2 in DRG neurons, leading to decreased central sensitization in the spinal cord. The findings may have implications for the development of novel therapeutic targets for the treatment of postoperative pain.

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.

Tiam1-mediated maladaptive plasticity underlying morphine tolerance and hyperalgesia.

Opioid pain medications, such as morphine, remain the mainstay for treating severe and chronic pain. Prolonged morphine use, however, triggers analgesic tolerance and hyperalgesia (OIH), which can last for a long period after morphine withdrawal. How morphine induces these detrimental side effects remains unclear. Here, we show that morphine tolerance and OIH are mediated by Tiam1-coordinated synaptic structural and functional plasticity in the spinal nociceptive network. Tiam1 is a Rac1 GTPase guanine nucleotide exchange factor (GEF) that promotes excitatory synaptogenesis by modulating actin cytoskeletal dynamics. We found that prolonged morphine treatment activated Tiam1 in the spinal dorsal horn and Tiam1 ablation from spinal neurons eliminated morphine antinociceptive tolerance and OIH. At the same time, the pharmacological blockade of Tiam1-Rac1 signaling prevented the development and reserved the established tolerance and OIH. Prolonged morphine treatment increased dendritic spine density and synaptic NMDA receptor (NMDAR) activity in spinal dorsal horn neurons, both of which required Tiam1. Furthermore, co-administration of the Tiam1 signaling inhibitor NSC23766 was sufficient to abrogate morphine tolerance in chronic pain management. These findings identify Tiam1-mediated maladaptive plasticity in the spinal nociceptive network as an underlying cause for the development and maintenance of morphine tolerance and OIH and provide a promising therapeutic target to reduce tolerance and prolong morphine use in chronic pain management.

Emerging Molecular and Synaptic Targets for the Management of Chronic Pain Caused by Systemic Lupus Erythematosus.

Patients with systemic lupus erythematosus (SLE) frequently experience chronic pain due to the limited effectiveness and safety profiles of current analgesics. Understanding the molecular and synaptic mechanisms underlying abnormal neuronal activation along the pain signaling pathway is essential for developing new analgesics to address SLE-induced chronic pain. Recent studies, including those conducted by our team and others using the SLE animal model (MRL/lpr lupus-prone mice), have unveiled heightened excitability in nociceptive primary sensory neurons within the dorsal root ganglia and increased glutamatergic synaptic activity in spinal dorsal horn neurons, contributing to the development of chronic pain in mice with SLE. Nociceptive primary sensory neurons in lupus animals exhibit elevated resting membrane potentials, and reduced thresholds and rheobases of action potentials. These changes coincide with the elevated production of TNFα and IL-1β, as well as increased ERK activity in the dorsal root ganglion, coupled with decreased AMPK activity in the same region. Dysregulated AMPK activity is linked to heightened excitability in nociceptive sensory neurons in lupus animals. Additionally, the increased glutamatergic synaptic activity in the spinal dorsal horn in lupus mice with chronic pain is characterized by enhanced presynaptic glutamate release and postsynaptic AMPA receptor activation, alongside the reduced activity of glial glutamate transporters. These alterations are caused by the elevated activities of IL-1β, IL-18, CSF-1, and thrombin, and reduced AMPK activities in the dorsal horn. Furthermore, the pharmacological activation of spinal GPR109A receptors in microglia in lupus mice suppresses chronic pain by inhibiting p38 MAPK activity and the production of both IL-1β and IL-18, as well as reducing glutamatergic synaptic activity in the spinal dorsal horn. These findings collectively unveil crucial signaling molecular and synaptic targets for modulating abnormal neuronal activation in both the periphery and spinal dorsal horn, offering insights into the development of analgesics for managing SLE-induced chronic pain.

Development and characterization of a Gucy2d-cre mouse to selectively manipulate a subset of inhibitory spinal dorsal horn interneurons.

Recent transcriptomic studies identified Gucy2d (encoding guanylate cyclase D) as a highly enriched gene within inhibitory dynorphin interneurons in the mouse spinal dorsal horn. To facilitate investigations into the role of the Gucy2d+ population in somatosensation, Gucy2d-cre transgenic mice were created to permit chemogenetic or optogenetic manipulation of this subset of spinal neurons. Gucy2d-cre mice created via CRISPR/Cas9 genomic knock-in were bred to mice expressing a cre-dependent reporter (either tdTomato or Sun1.GFP fusion protein), and the resulting offspring were characterized. Surprisingly, a much wider population of spinal neurons was labeled by cre-dependent reporter expression than previous mRNA-based studies would suggest. Although the cre-dependent reporter expression faithfully labeled ~75% of cells expressing Gucy2d mRNA in the adult dorsal horn, it also labeled a substantial number of additional inhibitory neurons in which no Gucy2d or Pdyn mRNA was detected. Moreover, cre-dependent reporter was also expressed in various regions of the brain, including the spinal trigeminal nucleus, cerebellum, thalamus, somatosensory cortex, and anterior cingulate cortex. Injection of AAV-CAG-FLEX-tdTomato viral vector into adult Gucy2d-cre mice produced a similar pattern of cre-dependent reporter expression in the spinal cord and brain, which excludes the possibility that the unexpected reporter-labeling of cells in the deep dorsal horn and brain was due to transient Gucy2d expression during early stages of development. Collectively, these results suggest that Gucy2d is expressed in a wider population of cells than previously thought, albeit at levels low enough to avoid detection with commonly used mRNA-based assays. Therefore, it is unlikely that these Gucy2d-cre mice will permit selective manipulation of inhibitory signaling mediated by spinal dynorphin interneurons, but this novel cre driver line may nevertheless be useful to target a broader population of inhibitory spinal dorsal horn neurons.

Inhibitory effect of acupoint electrostimulation with different layers and intensities on muscular inflammatory pain and spinal WDR neuron activity.

To observe the analgesic effects of different levels and intensities of electrical stimulation on the local acupoints in the pain source area and their impact on wide dynamic range (WDR) neurons in the spinal dorsal horn, in order to provide a basis for selecting appropriate parameters for electroacupuncture (EA) stimulation. Wistar rats were used in 3 parts of the experiment. Complete Freund's adjuvant was used to establish a model of inflammation-induced pain in the gastrocnemius muscle. After modeling, 6 rats were randomly selected for multi-channel extracellular electrophysiological recording of the electrical activity of WDR neurons, to determine the threshold for activating the A-component (Ta) and the C-component (Tc), which were used as the intervention intensities for skin transcutaneous electrical acupoint stimulation (TEAS) or EA. Thirty-six rats were randomly divided into normal , model , TEAS-Ta , TEAS-Tc, EA-Ta , and EA-Tc groups, with 6 rats in each group. In the pain source area , Ta or Tc intensity of TEAS or EA intervention at"Chengshan"(BL57) was performed for 30 min each time, once a day, for 3 consecutive days. A small animal pressure pain measurement instrument was used to measure the mechanical pressure pain threshold of the gastrocnemius muscle in rats, and the Von Frey filament was used to measure the mechanical pain threshold of the footpad. Thirteen rats were randomly selected to observe the immediate responsiveness of WDR neurons to Ta/Tc intensity of EA or TEAS in BL57. The thresholds of TEAS to activate WDR neuron A-component or C-component were (2.43±0.57) mA and (7.00±1.34) mA, respectively, while the thresholds for EA to activate muscle WDR neuron A-component or C-component were (0.72±0.34) mA and (1.58±0.35) mA, respectively. After injection of CFA into the gastrocnemius muscle, compared with the normal group both the mechanical pressure pain threshold of the gastrocnemius muscle and the mechanical pain threshold of the footpad of rats in the model group were significantly decreased (P<0.001). After TEAS-Ta, TEAS-Tc or EA-Ta intervention in the BL57, both the mechanical pressure pain threshold of the gastrocnemius muscle and the mechanical pain threshold of the footpad were significantly higher than those in the model group (P<0.05, P<0.001). Compared with the normal group, the electrical threshold for evoking WDR neuron C-component discharge was significantly decreased (P<0.001) in the model group, while increased after TEAS-Ta, TEAS-Tc, or EA-Ta intervention (P<0.01) compared with the model group. The evoked discharge frequency of muscle WDR neurons decreased significantly after immediate intervention with TEAS-Ta, TEAS-Tc, or EA-Ta (P<0.01, P<0.05). EA-Tc had no significant improvement on the evoked electrical activity of WDR neurons or pain behavior. TEAS-Ta, TEAS-Tc, or EA-Ta can all alleviate the local and footpad mechanical pain in rats with muscle inflammation and inhibit the responsiveness of WDR neurons, indicating that different intensities are required for analgesic effects at different levels of acupoints in the pain source area.

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.

Tau Accumulation in the Spinal Cord Contributes to Chronic Inflammatory Pain by Upregulation of IL-1β and BDNF

Microtubule-associated protein Tau is responsible for the stabilization of neuronal microtubules under normal physiological conditions. Much attention has been focused on Tau’s contribution to cognition, but little research has explored its role in emotions such as pain, anxiety, and depression. In the current study, we found a significant increase in the levels of p-Tau (Thr231), total Tau, IL-1β, and brain-derived neurotrophic factor (BDNF) on day 7 after complete Freund's adjuvant (CFA) injection; they were present in the vast majority of neurons in the spinal dorsal horn. Microinjection of Mapt-shRNA recombinant adeno-associated virus into the spinal dorsal cord alleviated CFA-induced inflammatory pain and inhibited CFA-induced IL-1β and BDNF upregulation. Importantly, Tau overexpression was sufficient to induce hyperalgesia by increasing the expression of IL-1β and BDNF. Furthermore, the activation of glycogen synthase kinase 3 beta partly contributed to Tau accumulation. These findings suggest that Tau in the dorsal horn could be a promising target for chronic inflammatory pain therapy.

Opioid-Induced Hyperalgesia and Tolerance Are Driven by HCN Ion Channels.

Prolonged exposure to opioids causes an enhanced sensitivity to painful stimuli (opioid-induced hyperalgesia, OIH) and a need for increased opioid doses to maintain analgesia (opioid-induced tolerance, OIT), but the mechanisms underlying both processes remain obscure. We found that pharmacological block or genetic deletion of HCN2 ion channels in primary nociceptive neurons of male mice completely abolished OIH but had no effect on OIT. Conversely, pharmacological inhibition of central HCN channels alleviated OIT but had no effect on OIH. Expression of C-FOS, a marker of neuronal activity, was increased in second-order neurons of the dorsal spinal cord by induction of OIH, and the increase was prevented by peripheral block or genetic deletion of HCN2, but block of OIT by spinal block of HCN channels had no impact on C-FOS expression in dorsal horn neurons. Collectively, these observations show that OIH is driven by HCN2 ion channels in peripheral nociceptors, while OIT is driven by a member of the HCN family located in the CNS. Induction of OIH increased cAMP in nociceptive neurons, and a consequent shift in the activation curve of HCN2 caused an increase in nociceptor firing. The shift in HCN2 was caused by expression of a constitutively active μ-opioid receptor (MOR) and was reversed by MOR antagonists. We identified the opioid-induced MOR as a six-transmembrane splice variant, and we show that it increases cAMP by coupling constitutively to Gs HCN2 ion channels therefore drive OIH, and likely OIT, and may be a novel therapeutic target for the treatment of addiction.

Defective autophagy contributes to bupivacaine-induced aggravation of painful diabetic neuropathy in db/db mice

Current evidence suggests that hyperactivated or impaired autophagy can lead to neuronal death. The effect of local anesthetics on painful diabetic neuropathy (PDN) and the role of autophagy in the above pathological process remain unclear, warranting further studies. So, PDN models were established by assessing the paw withdrawal threshold (PWT) and paw withdrawal latency (PWL) in leptin gene-mutation (db/db) mice. Wild type (WT) and PDN mice received intrathecal 0.75% bupivacaine or/with intraperitoneal drug treatment (rapamycin or bafilomycin A1). Subsequently, the PWT and PWL were measured to assess hyperalgesia at 6 h, 24 h, 30 h, and 48 h after intrathecal bupivacaine. Also, sensory nerve conduction velocity (SNCV) and motor nerve conduction velocity (MNCV) were measured before and 48 h after intrathecal bupivacaine treatment. The spinal cord tissue of L4-L6 segments and serum were harvested to evaluate the change of autophagy, oxidative stress, oxidative injury, and apoptosis. We found that bupivacaine induced the activation of autophagy but did not affect the pain threshold, SNCV and MNCV in WT mice at predefined time points. Conversely, bupivacaine lowered autophagosome generation and degradation, slowed SNCV and aggravated spinal dorsal horn neuron oxidative injury and hyperalgesia in PDN mice. The autophagy activator (rapamycin) could decrease spinal dorsal horn neuron oxidative injury, alleviate the alterations in SNCV and hyperalgesia in bupivacaine-treated PDN mice. Meanwhile, the autophagy inhibitor (bafilomycin A1) could exacerbate spinal dorsal horn neuron oxidative injury, the alterations in SNCV and hyperalgesia in bupivacaine-treated PDN mice. Our results showed that bupivacaine could induce defective autophagy, slowed SNCV and aggravate spinal dorsal horn neuron oxidative injury and hyperalgesia in PDN mice. Restoring autophagy may represent a potential therapeutic approach against nerve injury in PDN patients with local anesthesia and analgesia.

Constitutive KCC2 Cell- and Synapse-Specifically Regulates NMDA Receptor Activity in the Spinal Cord.

K+-Cl- cotransporter-2 (KCC2) critically controls neuronal chloride homeostasis and maintains normal synaptic inhibition by GABA and glycine. Nerve injury diminishes synaptic inhibition in the spinal cord via KCC2 impairment. However, how KCC2 regulates nociceptive input to spinal excitatory and inhibitory neurons remains elusive. Here, we show that basal GABA reversal potentials were significantly more depolarized in vesicular GABA transporter (VGAT)-expressing inhibitory neurons than those in vesicular glutamate transporter-2 (VGluT2)-expressing excitatory neurons in spinal cords of male and female mice. Strikingly, inhibiting KCC2 with VU0463271 increased currents elicited by puff NMDA and the NMDAR-mediated frequency of mEPSCs in VGluT2, but not in VGAT, dorsal horn neurons. Notably, VU0463271 had no effect on EPSCs monosynaptically evoked from the dorsal root in VGluT2 neurons. Furthermore, VU0463271 augmented α2δ-1-NMDAR interactions and their protein levels in spinal cord synaptosomes. In Cacna2d1 KO mice, VU0463271 had no effect on puff NMDA currents or the mEPSC frequency in dorsal horn neurons. Disrupting α2δ-1-NMDAR interactions with α2δ-1 C-terminus mimicking peptide diminished VU0463271-induced potentiation in the mEPSC frequency and puff NMDA currents in VGluT2 neurons. Additionally, intrathecal injection of VU0463271 reduced mechanical and thermal thresholds in wild-type mice, but not in Cacna2d1 KO mice. VU0463271-induced pain hypersensitivity in mice was abrogated by co-treatment with the NMDAR antagonist, pregabalin (an α2δ-1 inhibitory ligand), or α2δ-1 C-terminus mimicking peptide. Our findings suggest that KCC2 controls presynaptic and postsynaptic NMDAR activity specifically in excitatory dorsal horn neurons. KCC2 impairment preferentially potentiates nociceptive transmission between spinal excitatory interneurons via α2δ-1-bound NMDARs.Significance statementImpaired function of potassium-chloride cotransporter-2 (KCC2), a key regulator of neuronal inhibition, in the spinal cord plays a major role in neuropathic pain. This study unveils that KCC2 controls spinal nociceptive synaptic strength via NMDA receptors in a cell type- and synapse type-specific manner. KCC2 inhibition preferentially augments presynaptic and postsynaptic NMDA receptor activity in spinal excitatory interneurons via α2δ-1 (previously known as a calcium channel subunit). Importantly, spinal KCC2 impairment triggers pain hypersensitivity through α2δ-1-coupled NMDA receptors. These findings pinpoint the cell and molecular substrates for the reciprocal relationship between spinal synaptic inhibition and excitation in chronic neuropathic pain. Targeting both KCC2 and α2δ-1–NMDA receptor complexes could be an effective strategy in managing neuropathic pain conditions.

Specific inhibition of TET1 in the spinal dorsal horn alleviates inflammatory pain in mice by regulating synaptic plasticity

DNA demethylation mediated by ten-eleven translocation 1 (TET1) is a critical epigenetic mechanism in which gene expression is regulated via catalysis of 5-methylcytosine to 5-hydroxymethylcytosine. Previously, we demonstrated that TET1 is associated with the genesis of chronic inflammatory pain. However, how TET1 participates in enhanced nociceptive responses in chronic pain remains poorly understood. Here, we report that conditional knockout of Tet1 in dorsal horn neurons via intrathecal injection of rAAV-hSyn-Cre in Tet1fl/fl mice not only reversed the inflammation-induced upregulation of synapse-associated proteins (post-synaptic density protein 95 (PSD95) and synaptophysin (SYP)) in the dorsal horn but also ameliorated abnormalities in dendritic spine morphology and alleviated pain hypersensitivities. Pharmacological blockade of TET1 by intrathecal injection of a TET1-specific inhibitor—Bobcat 339—produced similar results, as did knockdown of Tet1 by intrathecal injection of siRNA. Thus, our data strongly suggest that increased TET1 expression during inflammatory pain upregulates the expression of multiple synapse-associated proteins and dysregulates synaptic morphology in dorsal horn neurons, suggesting that Tet1 may be a potential target for analgesic strategies.

Model-based analysis of subthreshold mechanisms of spinal cord stimulation for pain

Objective. Spinal cord stimulation (SCS) is a common treatment for chronic pain. For decades, SCS maximized overlap between stimulation-induced paresthesias and the patient’s painful areas. Recently developed SCS paradigms relieve pain at sub-perceptible amplitudes, yet little is known about the neural response to these new waveforms or their analgesic mechanisms of action. Therefore, in this study, we investigated the neural response to multiple forms of paresthesia-free SCS. Approach. We used computational modeling to investigate the neurophysiological effects and the plausibility of commonly proposed mechanisms of three paresthesia-free SCS paradigms: burst, 1 kHz, and 10 kHz SCS. Specifically, in C- and Aβ-fibers, we investigated the effects of different SCS waveforms on spike timing and activation thresholds, as well as how stochastic ion channel gating affects the response of dorsal column axons. Finally, we characterized membrane polarization of superficial dorsal horn neurons. Main results. We found that none of the SCS waveforms activate nor modulate spike timing in C-fibers. Spike timing was modulated in Aβ-fibers only at suprathreshold amplitudes. Ion channel stochasticity had little effect on Aβ-fiber activation thresholds but produced heterogeneous spike timings at suprathreshold amplitudes. Finally, local cells were preferentially polarized in their axon terminals, and the magnitude of this polarization was dependent on cellular morphology and position relative to the stimulation electrodes. Significance. Overall, the mechanisms of action of subparesthetic SCS remain unclear. Our results suggest that no SCS waveforms directly activate C-fibers, and modulation of spike timing is unlikely at subthreshold amplitudes. We conclude that potential subthreshold neuromodulatory effects of SCS on local cells are likely to be presynaptic in nature, as axons are preferentially depolarized during SCS.

Central and peripheral mechanism of MOTS-c attenuates pain hypersensitivity in a mice model of inflammatory pain

ABSTRACT Background Inflammatory pain is caused by damaged tissue or noxious stimuli, accompanied by the release of inflammatory mediators that often leads to severe hyperalgesia and allodynia with limited therapy options. Recently, a novel mitochondrial-derived peptide (named MOTS-c) was reported to regulate obesity, metabolic homeostasis and inflammatory response. The aim of this study was to investigate the effects of MOTS-c and its related regulatory mechanisms involved in inflammatory pain. Methods Male Kunming mice (8–10 weeks-old) were intraplantar injected with formalin, capsaicin, λ-Carrageenan and complete Freund adjuvant (CFA) to establish acute and chronic inflammatory pain. The effects of MOTS-c on the above inflammatory pain mice and its underlying mechanisms were examined by behavioral tests, quantitative polymerase chain reaction (qPCR), western blotting, enzyme linked immunosorbent assay (ELISA), immunohistochemistry (IHC) and immunofluorescence (IF). Results Behavioral experiments investigated the potential beneficial effects of MOTS-c on multiple acute and chronic inflammatory pain in mice. The results showed that MOTS-c treatment produced potent anti-allodynic effects in formalin-induced acute inflammatory pain, capsaicin-induced nocifensive behaviors and λ-Carrageenan/CFA-induced chronic inflammatory pain model. Further mechanistic studies revealed that central MOTS-c treatment significantly ameliorated CFA-evoked the release of inflammatory factors and activation of glial cells and neurons in the spinal dorsal horn. Moreover, peripheral MOTS-c treatment reduced CFA-evoked inflammatory responses in the surface structure of hindpaw skin, accompanied by inhibiting excitation of peripheral calcitonin gene-related peptide (CGRP) and P2X3 nociceptive neurons. Conclusions The present study indicates that MOTS-c may serve as a promising therapeutic target for inflammatory pain.

Ac4C Enhances the Translation Efficiency of Vegfa mRNA and Mediates Central Sensitization in Spinal Dorsal Horn in Neuropathic Pain.

N4-Acetylcytidine (ac4C), a highly conserved post-transcriptional machinery with extensive existence for RNA modification, plays versatile roles in various cellular processes and functions. However, the molecular mechanism by which ac4C modification mediates neuropathic pain remains elusive. Here, it is found that the enhanced ac4C modification promotes the recruitment of polysome in Vegfa mRNA and strengthens the translation efficiency following SNI. Nerve injury increases the expression of NAT10 and the interaction between NAT10 and Vegfa mRNA in the dorsal horn neurons, and the gain and loss of NAT10 function further confirm that NAT10 is involved in the ac4C modification in Vegfa mRNA and pain behavior. Moreover, the ac4C-mediated VEGFA upregulation contributes to the central sensitivity and neuropathic pain induced by SNI or AAV-hSyn-NAT10. Finally, SNI promotes the binding of HNRNPK in Vegfa mRNA and subsequently recruits the NAT10. The enhanced interaction between HNRNPK and NAT10 contributes to the ac4C modification of Vegfa mRNA and neuropathic pain. These findings suggest that the enhanced interaction between HNRNPK and Vegfa mRNA upregulates the ac4C level by recruiting NAT10 and contributes to the central sensitivity and neuropathic pain following SNI. Blocking this cascade may be a novel therapeutic approach in patients with neuropathic pain.

Role of Histone Deacetylase Inhibitor in Diabetic Painful Neuropathy.

Diabetic painful neuropathy (DPN) is one of the most detrimental complications of diabetes. Alterations in neuroinflammatory mediators play significant roles in the development of DPN. Infiltration of the neutrophils and monocyte/macrophages contributes substantial role in the degenerative process of the distal sciatic nerve by forming neutrophil extracellular traps (NETs) under diabetic condition. Citrullination of histones due to increase in protein arginine deiminase (PAD) enzyme activity under hyperglycemia may promote NET formation, which can further increase the cytokine production by activating macrophages and proliferation of neutrophils. This study reveals that the increase in histone deacetylases (HDAC) is crucial in DPN and inhibition of HDAC using HDAC inhibitor (HDACi) FK228 would suppress NETosis and alleviate diabetic nerve degeneration and pain. FK228, also known as romidepsin, is FDA approved for the treatment of cutaneous T-cell lymphoma yet the molecular mechanisms of this drug are not completely understood in DPN. In this study, type 2 diabetic (T2D) mice with pain were treated with HDACi, FK228 1mg/kg; I.P. 2 × /week for 3weeks. The results demonstrate that FK228 treatment can alleviate thermal hyperalgesia and mechanical allodynia significantly along with changes in the expression of HDACs in the dorsal root ganglia (DRG) and spinal cord dorsal horn neurons of diabetic animals. The results also indicate that FK228 treatment can alter the expression of neutrophil elastase (NE), extracellular or cell free DNA (cfDNA), citrullinated histone-3 (CitH3), PADI4, growth-associated protein (GAP)-43, and glucose transporter (GLUT)-4. Overall, this study suggests that FK228 could amend the expression of nerve regeneration markers and inflammatory mediators in diabetic animals and may offer an alternative treatment approach for DPN.

Optogenetic inhibition of spinal inhibitory neurons facilitates mechanical responses of spinal wide dynamic range neurons and causes mechanical hypersensitivity

Inhibitory interneurons in the spinal dorsal horn (DH) play a major role in regulating innocuous and noxious information. Reduction in inhibitory synaptic transmission is thought to contribute to the development of touch-evoked pain (allodynia), a common symptom of neuropathic pain. However, it is not fully understood how inhibitory neurons in the DH regulate sensory responses in surrounding neurons and modulate sensory transmission. In this study, we established a novel experimental method to analyze temporal activity of DH neurons during the optogenetically induced disinhibition state by combining extracellular recording and optogenetics. We investigated how specific and temporally restricted dysfunction of DH inhibitory neurons affected spinal neuronal activities evoked by cutaneous mechanical stimulation. In behavioral experiments, the specific and temporally restricted spinal optogenetic suppression of DH inhibitory neurons induced mechanical hypersensitivity. Furthermore, this manipulation enhanced the mechanical responses of wide dynamic range (WDR) neurons, which are important for pain transmission, in response to brush and von Frey stimulation but not in response to nociceptive pinch stimulation. In addition, we examined whether a neuropathic pain medication, mirogabalin, suppressed these optogenetically induced abnormal pain responses. We found that mirogabalin treatment attenuated the abnormal firing responses of WDR neurons and mechanical hypersensitivity. These results suggest that temporally restricted and specific reduction of spinal inhibitory neuronal activity facilitates the mechanical responses of WDR neurons, resulting in neuropathic-like mechanical allodynia which can be suppressed by mirogabalin. Our optogenetic methods could be useful for developing novel therapeutics for neuropathic pain.

Spinal-Specific Super Enhancer in Neuropathic Pain.

Dysfunctional gene expression in nociceptive pathways plays a critical role in the development and maintenance of neuropathic pain. Super enhancers (SEs), composed of a large cluster of transcriptional enhancers, are emerging as new players in the regulation of gene expression. However, whether SEs participate in nociceptive responses remains unknown. Here, we report a spinal-specific SE (SS-SE) that regulates chronic constriction injury (CCI)-induced neuropathic pain by driving Ntmt1 and Prrx2 transcription in dorsal horn neurons. Peripheral nerve injury significantly enhanced the activity of SS-SE and increased the expression of NTMT1 and PRRX2 in the dorsal horn of male mice in a bromodomain-containing protein 4 (BRD4)-dependent manner. Both intrathecal administration of a pharmacological BRD4 inhibitor JQ1 and CRISPR-Cas9-mediated SE deletion abolished the increased NTMT1 and PRRX2 in CCI mice and attenuated their nociceptive hypersensitivities. Furthermore, knocking down Ntmt1 or Prrx2 with siRNA suppressed the injury-induced elevation of phosphorylated extracellular-signal-regulated kinase (p-ERK) and glial fibrillary acidic protein (GFAP) expression in the dorsal horn and alleviated neuropathic pain behaviors. Mimicking the increase in spinal Ntmt1 or Prrx2 in naive mice increased p-ERK and GFAP expression and led to the genesis of neuropathic pain-like behavior. These results redefine our understanding of the regulation of pain-related genes and demonstrate that BRD4-driven increases in SS-SE activity is responsible for the genesis of neuropathic pain through the governance of NTMT1 and PRRX2 expression in dorsal horn neurons. Our findings highlight the therapeutic potential of BRD4 inhibitors for the treatment of neuropathic pain.SIGNIFICANCE STATEMENT SEs drive gene expression by recruiting master transcription factors, cofactors, and RNA polymerase, but their role in the development of neuropathic pain remains unknown. Here, we report that the activity of an SS-SE, located upstream of the genes Ntmt1 and Prrx2, was elevated in the dorsal horn of mice with neuropathic pain. SS-SE contributes to the genesis of neuropathic pain by driving expression of Ntmt1 and Prrx2 Both inhibition of SS-SE with a pharmacological BRD4 inhibitor and genetic deletion of SS-SE attenuated pain hypersensitivities. This study suggests an effective and novel therapeutic strategy for neuropathic pain.

Modulation of the spinal N13 SEP component by high- and low-frequency electrical stimulation. Experimental pain models matter

ObjectiveThe N13 component of somatosensory evoked potential (N13 SEP) represents the segmental response of cervical dorsal horn neurons. Neurophysiological studies in healthy participants showed that capsaicin-induced central sensitization causes an increase of the N13 SEP amplitude. Consequently, in human research, this spinal component may serve as a valuable readout of central sensitization.In this study, we wanted to verify if the sensitivity of the N13 SEP for detecting central sensitization is consistent across different experimental pain models inducing central sensitization and secondary hyperalgesia, namely high and low-frequency electrical stimulation (HFS and LFS). MethodsIn 18 healthy participants, we recorded SEP after bilateral ulnar nerve stimulation before and after secondary hyperalgesia was induced through HFS and LFS applied on the ulnar nerve territory of the hand of one side. The area of secondary hyperalgesia was mapped with a calibrated 128-mN pinprick probe, and the mechanical pain sensitivity with three calibrated 16–64-256-mN pinprick probes. ResultsAlthough both HFS and LFS successfully induced secondary hyperalgesia only LFS increased the amplitude of the N13 SEP. ConclusionsThese findings suggest that the sensitivity of the N13 SEP for detecting dorsal horn excitability changes may critically depend on the different experimental pain models. SignificanceOur results indicate that LFS and HFS could trigger central sensitization at the dorsal horn level through distinct mechanisms, however this still needs confirmation by replication studies.