Targeting spinal TRAF6 expression attenuates chronic visceral pain in adult rats with neonatal colonic inflammation

Background Irritable bowel syndrome is one of the most common gastrointestinal disorders. It is featured by abdominal pain in conjunction with altered bowel habits. However, the pathophysiology of the syndrome remains largely unknown. Tumor necrosis factor receptor-associated factor 6 (TRAF6) has been reported to be involved in neuropathic pain. The aim of this study was to investigate roles and mechanisms of TRAF6 in the chronic visceral hypersensitivity. Methods Visceral hypersensitivity was induced by neonatal colonic inflammation and was identified by colorectal distention. The protein level, RNA level, and cellular distribution of TRAF6 and its related molecules were detected with Western blot, quantitative polymerase chain reaction, and immunofluorescence. In vitro spinal cord slice recording technique was performed to determine the synaptic transmission activities. Results Neonatal colonic inflammation rats displayed visceral hypersensitivity at the age of six weeks. The expression of TRAF6 was obviously upregulated in spinal cord dorsal horn of neonatal colonic inflammation rats at the age of six weeks. Immunofluorescence study showed that TRAF6 was dominantly expressed in spinal astrocytes. Intrathecal injection of TRAF6 small interfering RNA (siRNA) significantly reduced the amplitude of spontaneous excitatory postsynaptic currents at the spinal dorsal horn level. Furthermore, knockdown of TRAF6 led to a significant downregulation of cystathionine β synthetase expression in the spinal dorsal horn of neonatal colonic inflammation rats. Importantly, intrathecal injection of TRAF6 siRNA remarkably alleviated visceral hypersensitivity of neonatal colonic inflammation rats. Conclusions Our results suggested that the upregulation of TRAF6 contributed to visceral pain hypersensitivity, which is likely mediated by regulating cystathionine β synthetase expression in the spinal dorsal horn. Our findings suggest that TRAF6 might act as a potential target for the treatment of chronic visceral pain in irritable bowel syndrome patients.

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Study on the Mechanism Underlying the Regulation of the NMDA Receptor Pathway in Spinal Dorsal Horns of Visceral Hypersensitivity Rats by Moxibustion

Evidence-based Complementary and Alternative Medicine ◽

10.1155/2016/3174608 ◽

2016 ◽

Vol 2016 ◽

pp. 1-11 ◽

Cited By ~ 2

Author(s):

L. D. Wang ◽

J. M. Zhao ◽

R. J. Huang ◽

L. Y. Tan ◽

Z. H. Hu ◽

...

Keyword(s):

Spinal Cord ◽

Irritable Bowel Syndrome ◽

Visceral Pain ◽

Intrathecal Injection ◽

Visceral Hypersensitivity ◽

Regulatory Function ◽

Visceral Hyperalgesia ◽

Spinal Dorsal ◽

Dorsal Horns ◽

Irritable Bowel

Visceral hypersensitivity is enhanced in irritable bowel syndrome (IBS) patients. Treatment of IBS visceral pain by moxibustion methods has a long history and rich clinical experience. In the clinic, moxibustion on the Tianshu (ST25) and Shangjuxu (ST37) acupoints can effectively treat bowel disease with visceral pain and diarrhea symptoms. To investigate the regulatory function of moxibustion on the Tianshu (ST25) and Shangjuxu (ST37) acupoints on spinal cord NR1, NR2B, and PKCεprotein and mRNA expression in irritable bowel syndrome (IBS) visceral hypersensitivity rats, we did some research. In the study, we found that moxibustion effectively relieved the IBS visceral hyperalgesia status of rats. Analgesic effect of moxibustion was similar to intrathecal injection of Ro 25-6981. The expression of NR1, NR2B, and PKCεin the spinal dorsal horns of IBS visceral hyperalgesia rats increased. Moxibustion on the Tianshu and Shangjuxu acupoints might inhibit the visceral hypersensitivity, simultaneously decreasing the expression of NR1, NR2B, and PKCεin spinal cord of IBS visceral hyperalgesia rats. Based on the above experimental results, we hypothesized NR1, NR2B, and PKCεof spinal cord could play an important role in moxibustion inhibiting the process of central sensitization and visceral hyperalgesia state.

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Upregulation of Spinal ASIC1 and NKCC1 Expression Contributes to Chronic Visceral Pain in Rats

Frontiers in Molecular Neuroscience ◽

10.3389/fnmol.2020.611179 ◽

2021 ◽

Vol 13 ◽

Author(s):

Yong-Chang Li ◽

Yuan-Qing Tian ◽

Yan-Yan Wu ◽

Yu-Cheng Xu ◽

Ping-An Zhang ◽

...

Keyword(s):

Synaptic Transmission ◽

Signaling Pathway ◽

Dorsal Horn ◽

Spinal Dorsal Horn ◽

Visceral Pain ◽

Intrathecal Injection ◽

Adult Rats ◽

Sodium Potassium ◽

Spinal Dorsal ◽

Whole Cell Patch Clamp

Aims: To determine whether acid-sensing ion channel 1 (ASIC1)–sodium-potassium-chloride cotransporter 1 (NKCC1) signaling pathway participates in chronic visceral pain of adult rats with neonatal maternal deprivation (NMD).Methods: Chronic visceral pain was detected by colorectal distension (CRD). Western blotting and Immunofluorescence were performed to detect the expression and location of ASIC1 and NKCC1. Whole-cell patch-clamp recordings were performed to record spinal synaptic transmission.Results: The excitatory synaptic transmission was enhanced and the inhibitory synaptic transmission was weakened in the spinal dorsal horn of NMD rats. ASIC1 and NKCC1 protein expression in the spinal dorsal horn was significantly up-regulated in NMD rats. Incubation of Amiloride reduced the amplitude of mEPSCs. Incubation of Bumetanide (BMT) increased the amplitude of mIPSCs. Intrathecal injection of ASIC1 or NKCC1 inhibitors reversed the threshold of CRD in NMD rats. Also, Amiloride treatment significantly reversed the expression of NKCC1 in the spinal dorsal horn of NMD rats.Conclusion: Our data suggest that the ASIC1-NKCC1 signaling pathway is involved in chronic visceral pain in NMD rats.

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Neonatal Colonic Inflammation Increases Spinal Transmission and Cystathionine β-Synthetase Expression in Spinal Dorsal Horn of Rats with Visceral Hypersensitivity

Frontiers in Pharmacology ◽

10.3389/fphar.2017.00696 ◽

2017 ◽

Vol 8 ◽

Cited By ~ 5

Author(s):

Liting Zhao ◽

Ying Xiao ◽

Rui-Xia Weng ◽

Xuelian Liu ◽

Ping-An Zhang ◽

...

Keyword(s):

Dorsal Horn ◽

Spinal Dorsal Horn ◽

Visceral Hypersensitivity ◽

Colonic Inflammation ◽

Spinal Dorsal

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Lack of Spinal Neuropeptide Y Is Involved in Mechanical Itch in Aged Mice

Frontiers in Aging Neuroscience ◽

10.3389/fnagi.2021.654761 ◽

2021 ◽

Vol 13 ◽

Author(s):

Huan Cui ◽

Wenliang Su ◽

Yan Cao ◽

Lulu Ma ◽

Guangyan Xu ◽

...

Keyword(s):

Spinal Cord ◽

Neuropeptide Y ◽

Dorsal Horn ◽

Spinal Dorsal Horn ◽

Elderly Population ◽

Intrathecal Injection ◽

The Elderly ◽

Aged Mice ◽

Spinal Dorsal ◽

Young Mice

Neuropeptide Y (NPY) signaling plays an essential role in gating the pruritic afferent information in the spinal cord. Recent studies revealed that the aging process down-regulated the expression of NPY in the central nervous system. We propose that the lack of spinal NPY may be involved in certain types of pruritus in the elderly population. This study was designed to investigate the role of NPY in aging-induced itch using the senile mouse model. The expression of NPY in the spinal dorsal horn was compared between young (2 months old) and aged (24 months old) mice. Western blotting and immunohistochemistry showed that the expression of NPY was significantly reduced in the spinal dorsal horn in aged mice. In addition, a neuronal maker of apoptosis, TUNEL, was detected in the NPY positive neurons only in the aged spinal cord. Behavioral assay indicated that light mechanical stimulus evoked significantly more scratching in the aged than in the young mice, whereas chemical-evoked itch and pain-related behaviors were not altered. Intrathecal injection of either NPY or LP-NPY, a NPY receptor 1 (NPY1R) agonist, significantly alleviated the mechanically evoked itch in aged mice without altering the responses to chemical pruritogens. Our study suggested that downregulation of spinal NPY in the aged mice might play a role in the higher incidence of the mechanically evoked itch than that in the young mice. Therapies targeting the NPY system might serve as a potential strategy for alleviating the pruritic symptoms among the elderly population.

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Sini-San Regulates the NO-cGMP-PKG Pathway in the Spinal Dorsal Horn in a Modified Rat Model of Functional Dyspepsia

Evidence-based Complementary and Alternative Medicine ◽

10.1155/2020/3575231 ◽

2020 ◽

Vol 2020 ◽

pp. 1-11 ◽

Cited By ~ 1

Author(s):

Zhenyu Wu ◽

Xiaofang Lu ◽

Shengsheng Zhang ◽

Chunyang Zhu

Keyword(s):

Gene Expression ◽

Dorsal Horn ◽

Functional Dyspepsia ◽

Rat Model ◽

Spinal Dorsal Horn ◽

Visceral Hypersensitivity ◽

Enzyme Linked Immunosorbent Assay ◽

Gastric Distention ◽

Real Time Quantitative Pcr ◽

Spinal Dorsal

The present study investigated the effect of Chinese medicine Sini-San (SNS) on visceral hypersensitivity in a rat model of functional dyspepsia (FD), and it explored related underlying mechanisms. The rat model of FD was developed by combining neonatal iodoacetamide (IA) treatment and adult tail-clamping. After SNS treatment, the behavior and electromyographic testing were performed to evaluate the visceromotor responses of rats to gastric distention. Immunofluorescence was used to detect the distribution of iNOS-positive cells in the spinal dorsal horn, while the real-time quantitative PCR and western blot were used for detection of the gene expression of c-fos, iNOS, and GABAb and protein levels of iNOS and GABAb in the spinal dorsal horn, respectively. The protein concentration of cGMP and PKG proteins in the spinal dorsal horn were quantified by enzyme-linked immunosorbent assay. In this study, SNS treatment significantly reduced the behavioral score and electromyographic response to graded intragastric distension pressure. The middle-dose of SNS treatment significantly reduced the distribution of iNOS-positive cells in the spinal dorsal horn of FD model rats. The gene expression of c-fos, iNOS, and GABAb and the protein contents of iNOS, GABAb, cGMP, and PKG in the spinal dorsal horn of FD model rats were restored to a normal level by middle-dose of SNS treatment. Our results suggest that Sini-San may alleviate the visceral hypersensitivity in FD model rats via regulation of the NO/cGMP/PKG pathway in the spinal dorsal horn.

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TWIK-Related Spinal Cord K+Channel Expression Is Increased in the Spinal Dorsal Horn after Spinal Nerve Ligation

Yonsei Medical Journal ◽

10.3349/ymj.2015.56.5.1307 ◽

2015 ◽

Vol 56 (5) ◽

pp. 1307 ◽

Cited By ~ 5

Author(s):

Hee Youn Hwang ◽

Enji Zhang ◽

Sangil Park ◽

Woosuk Chung ◽

Sunyeul Lee ◽

...

Keyword(s):

Spinal Cord ◽

Dorsal Horn ◽

Spinal Dorsal Horn ◽

Spinal Nerve ◽

Spinal Nerve Ligation ◽

K Channel ◽

Spinal Dorsal ◽

Channel Expression

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Metabotropic glutamate receptor 5 regulates excitability and Kv4.2-containing K+ channels primarily in excitatory neurons of the spinal dorsal horn

Journal of Neurophysiology ◽

10.1152/jn.01050.2010 ◽

2011 ◽

Vol 105 (6) ◽

pp. 3010-3021 ◽

Cited By ~ 29

Author(s):

Hui-Juan Hu ◽

Robert W. Gereau

Keyword(s):

Spinal Cord ◽

Dorsal Horn ◽

Spinal Dorsal Horn ◽

Neuronal Excitability ◽

Gabaergic Neurons ◽

Erk Signaling ◽

Metabotropic Glutamate ◽

Dorsal Horn Neurons ◽

Spinal Dorsal ◽

Excitatory Neurons

Metabotropic glutamate (mGlu) receptors play important roles in the modulation of nociception. Previous studies demonstrated that mGlu5 modulates nociceptive plasticity via activation of ERK signaling. We have reported recently that the Kv4.2 K+ channel subunit underlies A-type currents in spinal cord dorsal horn neurons and that this channel is modulated by mGlu5-ERK signaling. In the present study, we tested the hypothesis that modulation of Kv4.2 by mGlu5 occurs in excitatory spinal dorsal horn neurons. With the use of a transgenic mouse strain expressing enhanced green fluorescent protein (GFP) under control of the promoter for the γ-amino butyric acid (GABA)-synthesizing enzyme, glutamic acid decarboxylase 67 (GAD67), we found that these GABAergic neurons express less Kv4.2-mediated A-type current than non-GAD67-GFP neurons. Furthermore, the mGlu1/5 agonist, (R,S)-3,5-dihydroxyphenylglycine, had no modulatory effects on A-type currents or neuronal excitability in this subgroup of GABAergic neurons but robustly modulated A-type currents and neuronal excitability in non-GFP-expressing neurons. Immunofluorescence studies revealed that Kv4.2 was highly colocalized with markers of excitatory neurons, such as vesicular glutamate transporter 1/2, PKCγ, and neurokinin 1, in cultured dorsal horn neurons. These results indicate that mGlu5-Kv4.2 signaling is associated with excitatory dorsal horn neurons and suggest that the pronociceptive effects of mGlu5 activation in the spinal cord likely involve enhanced excitability of excitatory neurons.

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P2X3 Receptor in Primary Afferent Neurons Mediates the Relief of Visceral Hypersensitivity by Electroacupuncture in an Irritable Bowel Syndrome Rat Model

Gastroenterology Research and Practice ◽

10.1155/2020/8186106 ◽

2020 ◽

Vol 2020 ◽

pp. 1-10

Author(s):

Fang Zhang ◽

Zhe Ma ◽

Zhijun Weng ◽

Min Zhao ◽

Handan Zheng ◽

...

Keyword(s):

Irritable Bowel Syndrome ◽

Rat Model ◽

Visceral Pain ◽

Visceral Hypersensitivity ◽

Receptor Protein ◽

Resting Membrane Potential ◽

Drg Neurons ◽

Expression Levels ◽

Behavioral Studies ◽

Irritable Bowel

Background. Electroacupuncture (EA) has been confirmed effectiveness in the treatment of irritable bowel syndrome (IBS), and P2X3 receptors in the peripheral and central neurons participate in the acupuncture-mediated relief of the visceral pain in IBS. Objective. To reveal the neurobiological mechanism that P2X3 receptor of colonic primary sensory neurons in the dorsal root ganglia of the lumbosacral segment is involved in the alleviation of visceral hypersensitivity by EA in an IBS rat model. Methods. The IBS chronic visceral pain rat model was established according to the method of Al-Chaer et al. EA at the bilateral He-Mu points, including ST25 and ST37, was conducted for intervention. The behavioral studies, histopathology of colon, electrophysiology, immunofluorescence histochemistry, and real-time polymerase chain reaction assays were used to observe the role of P2X3 receptor in the colon and related DRG in relieving visceral hypersensitivity by EA. Results. EA significantly reduced the behavior scores of the IBS rats under different levels (20, 40, 60, 80 mmHg) of colorectal distention stimulation and downregulated the expression levels of P2X3 receptor protein and mRNA in colon and related DRG of the IBS rats. EA also regulated the electrical properties of the membranes, including the resting membrane potential, rheobase, and action potential of colon-associated DRG neurons in the IBS rats. Conclusion. EA can regulate the P2X3 receptor protein and mRNA expression levels in the colon and related DRG of IBS rats with visceral pain and then regulate the excitatory properties of DRG neurons.

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Synergistic Enhancement of Glutamate-Mediated Responses by Serotonin and Forskolin in Adult Mouse Spinal Dorsal Horn Neurons

Journal of Neurophysiology ◽

10.1152/jn.00423.2001 ◽

2002 ◽

Vol 87 (2) ◽

pp. 732-739 ◽

Cited By ~ 24

Author(s):

Guo-Du Wang ◽

Min Zhuo

Keyword(s):

Spinal Cord ◽

Nmda Receptors ◽

Dorsal Horn ◽

Spinal Dorsal Horn ◽

Adult Mouse ◽

Dorsal Horn Neurons ◽

Spinal Dorsal ◽

Afferent Fibers ◽

Primary Afferent Fibers ◽

Spinal Dorsal Horn Neurons

Glutamate is the major excitatory amino acid neurotransmitter in the CNS, including the neocortex, hippocampus, and spinal cord. Normal synaptic transmission is mainly mediated by glutamate AMPA and/or kainate receptors. Glutamate N-methyl-d-aspartate (NMDA) receptors are normally inactive and only activated when a sufficient postsynaptic depolarization is induced by the activity. Here we show that in sensory synapses of adult mouse, some synaptic responses (26.3% of a total of 38 experiments) between primary afferent fibers and dorsal horn neurons are almost completely mediated by NMDA receptors. Dorsal root stimulation did not elicit any detectable AMPA/kainate receptor-mediated responses in these synapses. Unlike young spinal cord, serotonin alone did not produce any long-lasting synaptic enhancement in adult spinal dorsal horn neurons. However, co-application of the adenylyl cyclase activator forskolin and serotonin (5-HT) produced long-lasting enhancement, including the recruitment of functional AMPA receptor-mediated responses. Calcium-sensitive, calmodulin-regulated adenylyl cyclases (AC1, AC8) are required for the enhancement. Furthermore the thresholds for generating action potential responses were decreased, and, in many cases, co-application of forskolin and 5-HT led to the generation of action potentials by previously subthreshold stimulation of primary afferent fibers in the presence of the NMDA receptor blocker 2-amino-5-phosphonovaleric acid. Our results suggest that pure NMDA synapses exist on sensory neurons in adult spinal cord and that they may contribute to functional sensory transmission. The synergistic recruitment of functional AMPA responses by 5-HT and forskolin provides a new cellular mechanism for glutamatergic synapses in mammalian spinal cord.

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