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Chronic visceral pain in patients with irritable bowel syndrome (IBS) has been difficult to treat effectively partially because its pathophysiology is not fully understood. Recent studies show that norepinephrine (NE) plays an important role in the development of visceral hypersensitivity. In this study, we designed to investigate the role of adrenergic signaling in visceral hypersensitivity induced by heterotypical intermittent stress (HIS). Abdominal withdrawal reflex scores (AWRs) used as visceral sensitivity were determined by measuring the visceromoter responses to colorectal distension. Colon-specific dorsal root ganglia neurons (DRGs) were labeled by injection of DiI into the colon wall and were acutely dissociated for whole-cell patch-clamp recordings. Blood plasma level of NE was measured using radioimmunoassay kits. The expression of β2-adrenoceptors was measured by western blotting. We showed that HIS-induced visceral hypersensitivity was attenuated by systemic administration of a β-adrenoceptor antagonist propranolol, in a dose-dependent manner, but not by a α-adrenoceptor antagonist phentolamine. Using specific β–adrenoceptor antagonists, HIS-induced visceral hypersensitivity was alleviated by β2 adrenoceptor antagonist but not by β1- or β3-adrenoceptor antagonist. Administration of a selective β2-adrenoceptor antagonist also normalized hyperexcitability of colon-innervating DRG neurons of HIS rats. Furthermore, administration of β-adrenoceptor antagonist suppressed sustained potassium current density (IK) without any alteration of fast-inactivating potassium current density (IA). Conversely, administration of NE enhanced the neuronal excitability and produced visceral hypersensitivity in healthy control rats, and blocked by β2-adrenoceptor antagonists. In addition, HIS significantly enhanced the NE concentration in the blood plasma but did not change the expression of β2-adrenoceptor in DRGs and the muscularis externa of the colon. The present study might provide a potential molecular target for therapy of visceral hypersensitivity in patents with IBS. [ABSTRACT FROM AUTHOR]

Patients with long-standing diabetes frequently demonstrate gastric hypersensitivity with an unknown mechanism. The present study was designed to investigate roles for nuclear factor-κB (NF-κB) and the endogenous H2S-producing enzyme cystathionine-β-synthetase (CBS) signaling pathways by examining cbs gene methylation status in adult rats with diabetes. Intraperitoneal injection of streptozotocin (STZ) produced gastric hypersensitivity in female rats in response to gastric balloon distention. Treatment with the CBS inhibitor aminooxyacetic acid significantly attenuated STZ-induced gastric hypersensitivity in a dose-dependent fashion. Aminooxyacetic acid treatment also reversed hyperexcitability of gastric-specific dorsal root ganglion (DRG) neurons labeled by the dye Dil in diabetic rats. Conversely, the H2S donor NaHS enhanced neuronal excitability of gastric DRG neurons. Expression of CBS and p65 were markedly enhanced in gastric DRGs in diabetic rats. Blockade of NF-κB signaling using pyrrolidine dithiocarbamate reversed the upregulation of CBS expression. Interestingly, STZ treatment led to a significant demethylation of CpG islands in the cbs gene promoter region, as determined by methylation-specific PCR and bisulfite sequencing. STZ treatment also remarkably downregulated the expression of DNA methyltransferase 3a and 3b. More importantly, STZ treatment significantly enhanced the ability of cbs to bind DNA at the p65 consensus site, as shown by chromatin immunoprecipitation assays. Our findings suggest that upregulation of cbs expression is attributed to cbs promoter DNA demethylation and p65 activation and that the enhanced interaction of the cbs gene and p65 contributes to gastric hypersensitivity in diabetes. This finding may guide the development and evaluation of new treatment modalities for patients with diabetic gastric hypersensitivity. [ABSTRACT FROM AUTHOR]

Background: Hydrogen sulfide (H2S), an endogenous gaseotransmitter/modulator, is becoming appreciated that it may be involved in a wide variety of processes including inflammation and nociception. However, the role and mechanism for H2S in nociceptive processing in trigeminal ganglion (TG) neuron remains unknown. The aim of this study is to investigate distribution of endogenous H2S synthesizing enzyme cystathionine-ß-synthetase (CBS) expression and role of H2S on excitability and voltage-gated potassium channels of TG neurons. Methods: Immunofluorescence studies were carried out to determine whether CBS was co-expressed in Kv1.1 or Kv1.4-positive TG neurons. Whole cell patch clamp recordings were employed on acutely isolated TG neurons from adult male Sprague Dawley rats (6-8 week old). von Frey filaments were used to examine the pain behavioral responses in rats following injection of sodium hydrosulfide. Results: In rat TG, 77.3±6.6% neurons were immunoreactive for CBS, 85.1±3.8% for Kv1.1 and 97.8±1.1% for Kv1.4. Double staining showed that all CBS labeled cells were Kv1.1 and Kv1.4 positive, but only 92.2±6.1% of Kv1.1 and 78.2±9.9% of Kv1.4 positive cells contained CBS. Application of H2S donor NaHS (250 µM) led to a significant depolarization of resting membrane potential recorded from TG neurons. NaHS application also resulted in a dramatic reduction in rheobase, hyperpolarization of action potential threshold, and a significant increase in the number of action potentials evoked at 2X and 3X rheobase stimulation. Under voltage-clamp conditions, TG neurons exhibited transient A-type (IA) and sustained outward rectifier K+ currents (IK). Application of NaHS did suppress IK density while did not change IA density of TG neurons (n=6). Furthermore, NaHS, a donor of hydrogen sulfide, produced a significant reduction in escape threshold in a dose dependent manner. Conclusion: These data suggest that endogenous H2S generating enzyme CBS was co-localized well with Kv1.1 and Kv1.4 in TG neurons and that H2S produces the mechanic pain and increases neuronal excitability, which might be largely mediated by suppressing IK density, thus identifying for the first time a specific molecular mechanism underlying pain and sensitization in TG. [ABSTRACT FROM AUTHOR]

Irritable bowel syndrome (IBS) is a prevalent functional gastrointestinal disease characterized by chronic visceral pain with a complex etiology and challenging treatment. Although accumulating evidence supports the involvement of central nervous system sensitization in the development of visceral pain, the precise molecular mechanisms remain incompletely understood. In this study, we highlight the critical regulatory role of lysine-specific demethylase 6B (KDM6B) in the anterior cingulate cortex (ACC) in chronic visceral pain. To simulate clinical IBS conditions, we utilized the neonatal maternal deprivation (NMD) mouse model. Our results demonstrated that NMD induced chronic visceral pain and anxiety-like behaviors in mice. Notably, the protein expression level of KDM6B significantly increased in the ACC of NMD mice, leading to a reduction in the expression level of H3K27me3. Immunofluorescence staining revealed that KDM6B primarily co-localizes with neurons in the ACC, with minimal presence in microglia and astrocytes. Injecting GSK-J4 (a KDM6B-specific inhibitor) into ACC of NMD mice, resulted in a significant alleviation in chronic visceral pain and anxiety-like behaviors, as well as a remarkable reduction in NR2B expression level. ChIP assay further indicated that KDM6B regulates NR2B expression by influencing the demethylation of H3K27me3. In summary, our findings underscore the critical role of KDM6B in regulating chronic visceral pain and anxiety-like behaviors in NMD mice. These insights provide a basis for further understanding the molecular pathways involved in IBS and may pave the way for targeted therapeutic interventions. [ABSTRACT FROM AUTHOR]

Aims: Irritable bowel syndrome (IBS) is a common functional gastrointestinal disorder, but its pathogenesis remains incompletely understood, particularly the involvements of central nervous system sensitization in colorectal visceral pain. Our study was to investigate whether the paraventricular thalamus (PVT) projected to the insular cortex (IC) to regulate colorectal visceral pain in neonatal colonic inflammation (NCI) mice and underlying mechanisms. Methods: We applied optogenetic, chemogenetic, or pharmacological approaches to manipulate the glutamatergicPVT‐IC pathway. Fiber photometry was used to assess neuronal activity. Electromyography activities in response to colorectal distension (CRD) were measured to evaluate the colorectal visceral pain. Results: NCI enhanced c‐Fos expression and calcium activity upon CRD in the ICGlu, and optogenetic manipulation of them altered colorectal visceral pain responses accordingly. Viral tracing indicated that the PVTGlu projected to the ICGlu. Optogenetic manipulation of PVTGlu changed colorectal visceral pain responses. Furthermore, selective optogenetic modulation of PVT projections in the IC influenced colorectal visceral pain, which was reversed by chemogenetic manipulation of downstream ICGlu. Conclusions: This study identified a novel PVT‐IC neural circuit playing a critical role in colorectal visceral pain in a mouse model of IBS. [ABSTRACT FROM AUTHOR]

Introduction: Neonatal stress disrupts brain development and increases the risk of neurological disorders later in life. However, the impact of neonatal stress on the development of the glymphatic system and susceptibility to Parkinson's disease (PD) remains largely unknown. Methods: Neonatal maternal deprivation (NMD) was performed on mice for 14 consecutive days to model chronic neonatal stress. Adeno‐associated virus expressing A53T‐α‐synuclein (α‐syn) was injected into the substantia nigra to establish PD model mice. Glymphatic activity was determined using in vivo magnetic resonance imaging, ex vivo fluorescence imaging and microplate assay. The transcription and expression of aquaporin‐4 (AQP4) and other molecules were evaluated by qPCR, western blotting, and immunofluorescence. Animal's responses to NMD and α‐syn overexpression were observed using behavioral tests. Results: Glymphatic activity was impaired in adult NMD mice. AQP4 polarization and platelet‐derived growth factor B (PDGF‐B) signaling were reduced in the frontal cortex and hippocampus of both young and adult NMD mice. Furthermore, exogenous α‐syn accumulation was increased and PD‐like symptoms were aggravated in adult NMD mice. Conclusion: The results demonstrated that NMD could disrupt the development of the glymphatic system through PDGF‐B signaling and increase the risk of PD later in life, indicating that alleviating neonatal stress could be beneficial in protecting the glymphatic system and reducing susceptibility to neurodegeneration. [ABSTRACT FROM AUTHOR]

Aims: Alzheimer's disease (AD) is a neurodegenerative disease characterized by progressive cognitive dysfunction and memory impairment. AD pathology involves protein acetylation. Previous studies have mainly focused on histone acetylation in AD, however, the roles of nonhistone acetylation in AD are less explored. Methods: The protein acetylation and expression levels were detected by western blotting and co‐immunoprecipitation. The stoichiometry of acetylation was measured by home‐made and site‐specific antibodies against acetylated‐CaM (Ac‐CaM) at K22, K95, and K116. Hippocampus‐dependent learning and memory were evaluated by using the Morris water maze, novel object recognition, and contextual fear conditioning tests. Results: We showed that calmodulin (CaM) acetylation is reduced in plasma of AD patients and mice. CaM acetylation and its target Ca2+/CaM‐dependent kinase II α (CaMKIIα) activity were severely impaired in AD mouse brain. The stoichiometry showed that Ac‐K22, K95‐CaM acetylation were decreased in AD patients and mice. Moreover, we screened and identified that lysine deacetylase 9 (HDAC9) was the main deacetylase for CaM. In addition, HDAC9 inhibition increased CaM acetylation and CaMKIIα activity, and hippocampus‐dependent memory in AD mice. Conclusions: HDAC9‐mediated CaM deacetylation induces memory impairment in AD, HDAC9, or CaM acetylation may become potential therapeutic targets for AD. [ABSTRACT FROM AUTHOR]

Background: Persistent pain is the most reported symptom in patients with rheumatoid arthritis (RA); however, effective and brief assessment tools are lacking. We validated the Chinese version of the Global Pain Scale (C-GPS) in Chinese patients with RA and proposed a short version of the C-GPS (s-C-GPS). Method: The study was conducted using a face-to-face questionnaire survey with a multicenter cross-sectional design from March to December 2019. Patients aged > 18 years who met the RA diagnostic criteria were included. Based on the classical test theory (CTT) and the item response theory (IRT), we assessed the validity and reliability of the C-GPS and the adaptability of each item. An s-C-GPS was developed using IRT-based computerized adaptive testing (CAT) analytics. Results: In total, 580 patients with RA (mean age, 51.04 ± 24.65 years; mean BMI, 22.36 ± 4.07 kg/m2), including 513 (88.4%) women, were included. Most participants lived in a suburb (49.3%), were employed (72.2%) and married (91.2%), reported 9–12 years of education (66.9%), and had partial medical insurance (57.8%). Approximately 88.1% smoked and 84.5% drank alcohol. Analysis of the CTT demonstrated that all items in the C-GPS were positively correlated with the total scale score, and the factor loadings of all these items were > 0.870. A significant positive relationship was found between the Visual Analog Scale (VAS) and the C-GPS. IRT analysis showed that discrimination of the C-GPS was between 2.271 and 3.312, and items 6, 8, 13, 14, and 16 provided a large amount of information. Based on the CAT and clinical practice, six items covering four dimensions were included to form the s-C-GPS, all of which had very high discrimination. The s-C-GPS positively correlated with the VAS. Conclusion: The C-GPS has good reliability and validity and can be used to evaluate pain in RA patients from a Chinese cultural background. The s-C-GPS, which contains six items, has good criterion validity and may be suitable for pain assessment in busy clinical practice. Trial registration: This cross-sectional study was registered in the Chinese Clinical Trial Registry (ChiCTR1800020343), granted on December 25, 2018. [ABSTRACT FROM AUTHOR]

Chronic visceral pain is a common symptom of irritable bowel syndrome (IBS). Exosomes are involved in the development of pain. Rab27a can mediate the release of exosomes. The purpose of this study is to investigate how Rab27a-mediated exosome secretion in the anterior cingulate cortex (ACC) regulates visceral hyperalgesia induced with neonatal maternal deprivation (NMD) in adult mice. The colorectal distension method was adopted to measure visceral pain. The BCA protein assay kit was applied to detect the exosome protein concentration. Western blotting, quantitative PCR, and immunofluorescence technique were adopted to detect the expression of Rab27a and the markers of exosomes. Exosomes extracted from ACC were more in NMD mice than in control (CON) mice. Injection of the exosome-specific inhibitor GW4869 in ACC attenuated colorectal visceral pain of NMD mice. Injection of NMD-derived exosomes produced colorectal visceral pain in CON mice. Rab27a was upregulated in ACC of NMD mice. Rab27a was highly expressed in ACC neurons of NMD mice, rather than astrocytes and microglia. Injection of Rab27a-siRNA reduced the release of exosomes and attenuated the colorectal visceral pain in NMD mice. This study suggested that overexpression of Rab27a increased exosome secretion in ACC neurons, thus contributing to visceral hyperalgesia in NMD mice. NEW & NOTEWORTHY This work demonstrated that the expression of Rab27a in the anterior cingulate cortex was upregulated, which mediated multivesicular bodies trafficking to the plasma membrane and led to the increased release of neuronal exosomes, thus contributing to colorectal visceral pain in neonatal maternal deprivation (NMD) mice. Blocking the release of exosomes or downregulation of Rab27a could alleviate colorectal visceral pain in NMD mice. These data may provide a promising strategy for the treatment of visceral pain in irritable bowel syndrome patients. [ABSTRACT FROM AUTHOR]

Supplemental Digital Content is Available in the Text. Wu et al. report that miR-1306-3p might be an endogenous ligand to activate P2X3 receptors, thus enhancing dorsal root ganglion neuron excitability and contributing to visceral pain. Mounting evidence indicates that microRNAs (miRNAs) play critical roles in various pathophysiological conditions and diseases, but the physiological roles of extracellular miRNAs on the disease-related ion channels remain largely unknown. Here, we showed that miR-1306-3p evoked action potentials and induced inward currents of the acutely isolated rat dorsal root ganglion (DRG) neurons. The miR-1306-3p–induced effects were significantly inhibited by A317491, a potent inhibitor of the P2X3 receptor (P2X3R), or disappeared after the knockdown of P2X3Rs in DRG neurons. We further identified R180, K315, and R52 as the miR-1306-3p interaction sites on the extracellular domain of P2X3Rs, which were distinct from the orthosteric ATP-binding sites. Intrathecal injection of miR-1306-3p produced visceral pain but not somatic pain in normal control rats. Conversely, intrathecal application of a miR-1306-3p antagomir and A317491 significantly alleviated visceral pain in a rat model of chronic visceral pain. Together, our findings suggest that miR-1306-3p might function as an endogenous ligand to activate P2X3Rs, eventually leading to chronic visceral pain. [ABSTRACT FROM AUTHOR]

Aims: Gastric hypersensitivity (GHS) is a characteristic pathogenesis of functional dyspepsia (FD). DNA methyltransferase 1 (DNMT1) and acid‐sensing ion channel 1 (ASIC1) are associated with GHS induced by prenatal maternal stress (PMS). The aim of this study was to investigate the mechanism of DNMT1 mediating the analgesic effect of folic acid (FA) on PMS‐induced GHS. Methods: GHS was quantified by electromyogram recordings. The expression of DNMT1, DNMT3a, DNMT3b, and ASIC1 were detected by western blot, RT‐PCR, and double‐immunofluorescence. Neuronal excitability and proton‐elicited currents of dorsal root ganglion (DRG) neurons were determined by whole‐cell patch clamp recordings. Results: The expression of DNMT1, but not DNMT3a or DNMT3b, was decreased in DRGs of PMS rats. FA alleviated PMS‐induced GHS and hyperexcitability of DRG neurons. FA also increased DNMT1 and decreased ASIC1 expression and sensitivity. Intrathecal injection of DNMT1 inhibitor DC‐517 attenuated the effect of FA on GHS alleviation and ASIC1 downregulation. Overexpression of DNMT1 with lentivirus not only rescued ASIC1 upregulation and hypersensitivity, but also alleviated GHS and hyperexcitability of DRG neurons induced by PMS. Conclusions: These results indicate that increased DNMT1 contributes to the analgesic effect of FA on PMS‐induced GHS by reducing ASIC1 expression and sensitivity. [ABSTRACT FROM AUTHOR]

Supplemental Digital Content is Available in the Text. Paraventricular hypothalamic CaMKIIα-positive neurons input to ventral of lateral septal CaMKIIα-positive neurons controls chronic visceral pain. Irritable bowel syndrome is a functional gastrointestinal disorder characterized by chronic visceral pain with complex etiology and difficult treatment. Accumulated evidence has confirmed that the sensitization of the central nervous system plays an important role in the development of visceral pain, whereas the exact mechanisms of action of the neural pathways remain largely unknown. In this study, a distinct neural circuit was identified from the paraventricular hypothalamic (PVH) to the ventral of lateral septal (LSV) region. This circuit was responsible for regulating visceral pain. In particular, the data indicated that the PVH CaMKIIα-positive neurons inputs to the LSV CaMKIIα-positive neurons were only activated by colorectal distention rather than somatic stimulations. The PVH-LSV CaMKIIα+ projection pathway was further confirmed by experiments containing a viral tracer. Optogenetic inhibition of PVH CaMKIIα+ inputs to LSV CaMKIIα-positive neurons suppressed visceral pain, whereas selective activation of the PVH-LSV CaMKIIα+ projection evoked visceral pain. These findings suggest the critical role of the PVH-LSV CaMKIIα+ circuit in regulating visceral pain. [ABSTRACT FROM AUTHOR]