Thalamic Nucleus Reuniens Glutamatergic Neurons Mediate Colorectal Visceral Pain in Mice via 5-HT2B Receptors.

Irritable bowel syndrome (IBS) is a common functional bowel disorder characterized by abdominal pain and visceral hypersensitivity. Reducing visceral hypersensitivity is the key to effectively relieving abdominal pain in IBS. Increasing evidence has confirmed that the thalamic nucleus reuniens (Re) and 5-hydroxytryptamine (5-HT) neurotransmitter system play an important role in the development of colorectal visceral pain, whereas the exact mechanisms remain largely unclear. In this study, we found that high expression of the 5-HT2B receptors in the Re glutamatergic neurons promoted colorectal visceral pain. Specifically, we found that neonatal maternal deprivation (NMD) mice exhibited visceral hyperalgesia and enhanced spontaneous synaptic transmission in the Re brain region. Colorectal distension (CRD) stimulation induced a large amount of c-Fos expression in the Re brain region of NMD mice, predominantly in glutamatergic neurons. Furthermore, optogenetic manipulation of glutamatergic neuronal activity in the Re altered colorectal visceral pain responses in CON and NMD mice. In addition, we demonstrated that 5-HT2B receptor expression on the Re glutamatergic neurons was upregulated and ultimately promoted colorectal visceral pain in NMD mice. These findings suggest a critical role of the 5HT2B receptors on the Re glutamatergic neurons in the regulation of colorectal visceral pain.

Rab27a-mediated exosome secretion in anterior cingulate cortex contributes to colorectal visceral pain in adult mice with neonatal maternal deprivation.

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.

Folic acid attenuates chronic visceral pain by reducing clostridiales abundance and hydrogen sulfide production.

Irritable bowel syndrome (IBS) related chronic visceral pain affects 20% of people worldwide. The treatment options are very limited. Although the scholarly reviews have appraised the potential effects of the intestinal microbiota on intestinal motility and sensation, the exact mechanism of intestinal microbiota in IBS-like chronic visceral pain remains largely unclear. The purpose of this study is to investigate whether Folic Acid (FA) attenuated visceral pain and its possible mechanisms. Chronic visceral hyperalgesia was induced in rats by neonatal colonic inflammation (NCI). 16S rDNA analysis of fecal samples from human subjects and rats was performed. Patch clamp recording was used to determine synaptic transmission of colonic-related spinal dorsal horn. Alpha diversity of intestinal flora was increased in patients with IBS, as well as the obviously increased abundance of Clostridiales order (a main bacteria producing hydrogen sulfide). The hydrogen sulfide content was positive correlation with visceral pain score in patients with IBS. Consistently, NCI increased Clostridiales frequency and hydrogen sulfide content in feces of adult rats. Notably, the concentration of FA was markedly decreased in peripheral blood of IBS patients compared with non-IBS human subjects. FA supplement alleviated chronic visceral pain and normalized the Clostridiales frequency in NCI rats. In addition, FA supplement significantly reduced the frequency of sEPSCs of neurons in the spinal dorsal horn of NCI rats. Folic Acid treatment attenuated chronic visceral pain of NCI rats through reducing hydrogen sulfide production from Clostridiales in intestine.

A paraventricular hypothalamic nucleus input to ventral of lateral septal nucleus controls chronic visceral pain.

ABSTRACT: 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.

Identification of a Glutamatergic Claustrum-Anterior Cingulate Cortex Circuit for Visceral Pain Processing.

Chronic visceral pain is a major challenge for both patients and health providers. Although the central sensitization of the brain is thought to play an important role in the development of visceral pain, the detailed neural circuits remain largely unknown. Using a well-established chronic visceral hypersensitivity model induced by neonatal maternal deprivation (NMD) in male mice, we identified a distinct pathway whereby the claustrum (CL) glutamatergic neuron projecting to the anterior cingulate cortex (ACC) is critical for visceral pain but not for CFA-evoked inflammatory pain. By a combination of in vivo circuit-dissecting extracellular electrophysiological approaches and visceral pain related electromyographic (EMG) recordings, we demonstrated that optogenetic inhibition of CL glutamatergic activity suppressed the ACC neural activity and visceral hypersensitivity of NMD mice whereas selective activation of CL glutamatergic activity enhanced the ACC neural activity and evoked visceral pain of control mice. Further, optogenetic studies demonstrate a causal link between such neuronal activity and visceral pain behaviors. Chemogenetic activation or inhibition of ACC neural activities reversed the effects of optogenetic manipulation of CL neural activities on visceral pain responses. Importantly, molecular detection showed that NMD significantly enhances the expression of NMDA receptors and activated CaMKIIα in the ACC postsynaptic density (PSD) region. Together, our data establish a functional role for CL→ACC glutamatergic neurons in gating visceral pain, thus providing a potential treatment strategy for visceral pain.SIGNIFICANCE STATEMENT Studies have shown that sensitization of anterior cingulate cortex (ACC) plays an important role in chronic pain. However, it is as yet unknown whether there is a specific brain region and a distinct neural circuit that helps the ACC to distinguish visceral and somatic pain. The present study demonstrates that claustrum (CL) glutamatergic neurons maybe responding to colorectal distention (CRD) rather than somatic stimulation and that a CL glutamatergic projection to ACC glutamatergic neuron regulates visceral pain in mice. Furthermore, excessive NMDA receptors and overactive CaMKIIα in the ACC postsynaptic density (PSD) region were observed in mice with chronic visceral pain. Together, these findings reveal a novel neural circuity underlying the central sensitization of chronic visceral pain.

Overexpression of GRK6 alleviates chronic visceral hypersensitivity through downregulation of P2Y6 receptors in anterior cingulate cortex of rats with prenatal maternal stress.

AIMS: Visceral hypersensitivity is a major clinic symptom in patients with irritable bowel syndrome (IBS). Anterior cingulate cortex (ACC) is involved in processing the information of pain. Both G protein-coupled receptor kinase 6 (GRK6) and P2Y purinoceptor 6 (P2Y6) are associated with neuroinflammation and pathological pain. The aim of this study was to investigate the interaction between GRK6 and P2Y6 in ACC in the development of visceral hypersensitivity of adult offspring rats with prenatal maternal stress (PMS). METHODS: Visceral hypersensitivity was quantified by abdominal withdrawal reflex threshold to colorectal distension (CRD). The expression and cellular distribution of GRK6 and P2Y6 were determined by Western blotting, qPCR, and fluorescence immunohistochemistry. Co-immunoprecipitation was used to evaluate the interaction between GRK6 and P2Y6. RESULTS: The mRNA and protein levels of GRK6 were significantly decreased in ACC of PMS rats. The injection of GRK6 overexpression virus significantly attenuated visceral hypersensitivity of PMS rats. P2Y6's mRNA level, protein level, and ratio of membrane protein over total protein expression was markedly increased in PMS rats. P2Y6 antagonist MRS2578 microinjection reversed visceral hypersensitivity of PMS rats. GRK6 overexpression significantly reduced P2Y6's expression in membrane proteins and P2Y6's ratio of membrane protein over total protein expression. CONCLUSIONS: These results indicate that decreased GRK6 leads to the accumulation of P2Y6 at neuron membrane in ACC, thereby contributing to visceral hypersensitivity of PMS rats.

Targeting GATA1 and p2x7r Locus Binding in Spinal Astrocytes Suppresses Chronic Visceral Pain by Promoting DNA Demethylation.

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

Upregulation of spinal ASIC1 by miR-485 mediates enterodynia in adult offspring rats with prenatal maternal stress.

AIMS: Irritable bowel syndrome (IBS) is a common functional gastrointestinal disease characterized by abdominal pain. Our recent study has shown that the acid-sensitive ion channel 1 (ASIC1) in dorsal root ganglion (DRG) is involved in stomachache of adult offspring rats subjected with prenatal maternal stress (PMS). MiR-485 is predicted to target the expression of ASIC1. The aim of the present study was designed to determine whether miR-485/ASIC1 signaling participates in enterodynia in the spinal dorsal horn of adult offspring rats with PMS. METHODS: Enterodynia was measured by colorectal distension (CRD). Western blotting, qPCR, and in situ hybridization were performed to detect the expression of ASICs and related miRNAs. Spinal synaptic transmission was also recorded by patch clamping. RESULTS: PMS offspring rats showed that spinal ASIC1 protein expression and synaptic transmission were significantly enhanced. Administration of ASICs antagonist amiloride suppressed the synaptic transmission and enterodynia. Besides, PMS induced a significant reduction in the expression of miR-485. Upregulating the expression markedly attenuated enterodynia, reversed the increase in ASIC1 protein and synaptic transmission. Furthermore, ASIC1 and miR-485 were co-expressed in NeuN-positive spinal dorsal horn neurons. CONCLUSIONS: Overall, these data suggested that miR-485 participated in enterodynia in PMS offspring, which is likely mediated by the enhanced ASIC1 activities.

Overexpression of Purinergic P2X4 Receptors in Hippocampus Rescues Memory Impairment in Rats with Type 2 Diabetes.

Purinergic receptors have been reported to be involved in brain disorders. In this study, we explored their roles and mechanisms underlying the memory impairment in rats with type 2 diabetes mellitus (T2DM). T2DM rats exhibited a worse performance in the T-maze and Morris water maze (MWM) than controls. Microglia positive for P2X purinoceptor 4 (P2X4R) in the hippocampus were reduced and activated microglia were increased in T2DM rats. Long Amplicon PCR (LA-PCR) showed that DNA amplification of the p2x4r gene in the hippocampus was lower in T2DM rats. Minocycline significantly reduced the number of activated microglia and the mean distance traveled by T2DM rats in the MWM. Most importantly, P2X4R overexpression suppressed the activated microglia and rescued the memory impairment of T2DM rats. Overall, T2DM led to excessive activation of microglia in the hippocampus, partly through the DNA damage-mediated downregulation of P2X4Rs, thus contributing to memory impairment.

The expression of histone methyltransferases and distribution of selected histone methylations in testes of yak and cattle-yak hybrid.

Interspecies hybridization exists widely in nature and plays an important role in animal evolution and adaptation. It is commonly recognized that male offspring of interspecies hybrid are often sterile, which presents a crucial way of reproductive isolation. Currently, the mechanisms underlying interspecies hybrid male sterility are not well understood. Cattle-yak, progeny of yak (Bos grunniens) and cattle (Bos taurus) cross, is a unique animal model for investigating hybrid male sterility. Because histone modifications are vital for spermatogenesis, herein, we examined expressions of histone methyltransferases (HMTs) and distributions of histone methylations in the yak and cattle-yak testis. Histological examination of seminiferous tubules revealed that gonocytes and spermatocytes were established normally, however, spermatogenesis was arrested at the meiosis phase began at 10 months after birth in the hybrids. SUV420H1 was the only HMT examined showing a significant enrichment in cattle-yak testes at 3 months. Relative expressions of MLL5, SETDB1 and SUV420H1 were increased while SETDB2 and EZH2 were decreased in cattle-yak testes at 10 months. Relative concentrations of MLL5 and SUV420H1 were again increased while EHMT2 and PRDM9 expressions were decreased at 24 months. Immunofluorescent detection of selected histone methylations in cross-sections of testicular tissues or meiotic chromosomes demonstrated that depletion of H3K4me3 and significant enrichment of H3K27me3 and H4K20me3 were observed in Sertoli cells of cattle-yak. Levels and localizations of H3K4me3, H3K9me1, H3K9me3 and H4K20me3 were strikingly different in meiotic chromosomes of cattle-yak spermatocytes. These results highlighted the potential roles of histone methylations in spermatogenic failure and hybrid male sterility.

lncRNA ANRIL Ameliorates Oxygen and Glucose Deprivation (OGD) Induced Injury in Neuron Cells via miR-199a-5p/CAV-1 Axis.

Ischemia stroke is one of the leading causes of death and disability in the world. Long non-coding RNA ANRIL has been reported to play an important role in ischemic injury. In this study, we aim to explore the mechanism by which ANRIL exhibits protective effect. Middle cerebral artery occlusion mouse models were applied and infarction areas were assessed by TTC assay. The expression of ANRIL and miR-199a-5p were determined by qPCR. Oxygen and glucose deprivation treatment was applied to mimic in vitro ischemia injury in N-2a cells. The levels of BCL-2, BAX, MEK, ERK, CAV-1 were determined by western blot. Cell viability were assessed by MTT assay. The direct interaction among miR-199a-5p and ANRIL, miR-199a-5p and CAV-1 were demonstrated by dual Luciferase report assay. ANRIL and miR-199a-5p expression were changed in both in vivo and in vitro ischemia model. Overexpression of ANRIL or inhibition of miR-199a-5p could protect cells against ischemia induced injury by elevating cell viability through CAV-1 mediated MEK/ERK pathway. miR-199a-5p attenuated CAV-1 expression by direct targeting. ANRIL competitively interacted with miR-199a-5p in N-2a cells, leading to a de-repression of CAV-1. ANRIL protects N-2a cells against ischemia induced injury by elevated CAV-1 by competitively interacting with miR-199a-5p, thus activating MEK/ERK pathway and elevating cell viability.

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

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.

Integrated chemical and transcriptomic analyses unveils synthetic characteristics of different medicinal root parts of Angelica sinensis.

Objective: Why are different medicinal parts including heads, bodies and tails of Angelicae Sinensis Radix (ASR) distinct in pharmaceutical activities? Here we explored their discrepancy in chemical constituents and transcriptome. Methods: ASR were separated into three medicinal parts: heads (rootstocks with petiole traces of ASR), bodies (taproots of ASR) and tails (lateral roots of ASR), and chemical and transcriptomic analyses were conducted simultaneously. Results: High performance liquid chromatography (HPLC) fingerprint results showed that five widely used active ingredients (ferulic acid, senkyunolide H, senkyunolide A, n-butylphathlide, and ligustilide) were distributed unevenly in the three ASR medicinal parts. Partial least squares-discriminant analysis (PLS-DA) demonstrated that the heads can be differentiated from the two other root parts due to different amounts of the main components. However, the content of ferulic acid (a main quality marker) was significantly higher in tails than in the heads and bodies. The transcriptome analysis found that 25,062, 10,148 and 29,504 unigenes were specifically expressed in the heads, bodies and tails, respectively. WGCNA analysis identified 17 co-expression modules, which were constructed from the 19,198 genes in the nine samples of ASR. Additionally, we identified 28 unigenes involved in two phenylpropanoid biosynthesis (PB) pathways about ferulic acid metabolism pathways, of which 17 unigenes (60.7%) in the PB pathway were highly expressed in the tails. The expression levels of PAL, C3H, and CQT transcripts were significantly higher in the tails than in other root parts. RT-qPCR analysis confirmed that PAL, C3H, and CQT genes were predominantly expressed in the tail parts, especially PAL, whose expression was more than doubled as compared with that in other root parts. Conclusion: Chemical and transcriptomic analyses revealed the distribution contents and pivotal transcripts of the ferulic acid biosynthesis-related pathways. The spatial gene expression pattern partially explained the discrepancy of integral medicinal activities of three medicinal root parts.

Sample Size for Assessing Agreement between Two Methods of Measurement by Bland-Altman Method.

The Bland-Altman method has been widely used for assessing agreement between two methods of measurement. However, it remains unsolved about sample size estimation. We propose a new method of sample size estimation for Bland-Altman agreement assessment. According to the Bland-Altman method, the conclusion on agreement is made based on the width of the confidence interval for LOAs (limits of agreement) in comparison to predefined clinical agreement limit. Under the theory of statistical inference, the formulae of sample size estimation are derived, which depended on the pre-determined level of α, ß, the mean and the standard deviation of differences between two measurements, and the predefined limits. With this new method, the sample sizes are calculated under different parameter settings which occur frequently in method comparison studies, and Monte-Carlo simulation is used to obtain the corresponding powers. The results of Monte-Carlo simulation showed that the achieved powers could coincide with the pre-determined level of powers, thus validating the correctness of the method. The method of sample size estimation can be applied in the Bland-Altman method to assess agreement between two methods of measurement.

[The value of magnifying endoscopy combined with human telomerase reverse transcriptase in diagnosing precancerous gastric lesions].

OBJECTIVE: To explore the diagnostic value of magnifying endoscopy and human telomerase reverse transcriptase (hTERT) in identifying precancerous lesions of gastric mucosa. METHODS: 154 patients with upper digestive symptoms were included in the study. They were examined by the same senior doctor using magnifying endoscopy, and real-time diagnosis was made during the examination. Forceps biopsies were taken for pathological examination and were examined the expression of human telomerase reverse transcriptase mRNA (hTERT mRNA) by real-time quantitative PCR. The morphology of gastric pits under magnifying endoscopy was classified as follows: type A: round spot pits, type B: linear, type C: sparsely and thickly linear, typed D: patchy, and type E: villous. RESULTS: The divergence of intestinal metaplasia had significant differences implications among the gastric pit patterns from B to E under magnifying endoscopy (chi(2) = 17.58, P < 0.05). The more sever the intestinal metaplasia, the higher the pattern scale of the gastric pit (Pearson = 0.531, P < 0.05). The degree of dysplasia had a parallel relationship with the gastric pit patterns changed from C to E (chi(2) = 10.256, P < 0.05, Pearson = 0.549, P < 0.05). The expressions of hTERT mRNA of mucosa had significant differences among the gastric pit patterns B, C, D and E (F = 3.274, P < 0.05); If the expression of hTERT mRNA were taken as diagnostic criteria of gastric cancer, 65% of pit type E would be considered as gastric cancer which were otherwise negative by pathology. CONCLUSIONS: The micro-structural changes of gastric mucosa under magnifying endoscopy can reflect the severity of gastric disease, hTERT is a very important marker for diagnosis of gastric carcinoma. It is useful to predict the malignant change of gastric precancerous lesions in patients by following up high expression of hTERT mRNA combined with pit type E diagnosed by magnifying endoscopy.

[Practice and experience of the scientific research management].

The practice and experience of the scientific research management in Nanjing General Hospital from 1997 to 2002 is summarized in this review. By presenting the achievement gained in the past 6 years, the author prove to the scientific research managers that the effective manage thought is the key of scientific research management, and also expatriate the experience of the key points management in the management of scientific research subjects.