Stress-induced Visceral Hyperalgesia Research Articles

Photobiomodulation improves acute restraint stress-induced visceral hyperalgesia in rats

The purpose of this study is to explore the potential application of photobiomodulation to irritable bowel syndrome. We established the following experimental groups: the Non-Stress + Sham group, which consisted of rats that were not restrained and were only subjected to sham irradiation; the Stress + Sham group, which underwent 1 hour of restraint stress followed by sham irradiation; and the Stress + Laser group, which was subjected to restraint stress and percutaneous laser irradiation bilaterally on the L6 dorsal root ganglia for 5 minutes each. The experiment was conducted twice, with three and two laser conditions examined. Following laser irradiation, a barostat catheter was inserted into the rat’s colon. After a 30-minute acclimatization period, the catheter was inflated to a pressure of 60 mmHg, and the number of abdominal muscle contractions was measured over a 5-minute period. The results showed that photobiomodulation significantly suppressed the number of abdominal muscle contractions at average powers of 460, 70, and 18 mW. However, no significant suppression was observed at average powers of 1 W and 3.5 mW. This study suggests that photobiomodulation can alleviate visceral hyperalgesia induced by restraint stress, indicating its potential applicability to irritable bowel syndrome.

Altered gene expression signatures by calcitonin gene-related peptide promoted mast cell activity in the colon of stress-induced visceral hyperalgesia mice.

Calcitonin gene-related peptide (CGRP) is possibly involved in recruitment of mucosal mast cells (MCs) in the gut that may be associated with the development of irritable bowel syndrome (IBS), but the role of CGRP on the activation of MCs is still unknown. Using RNA sequencing (RNA-seq), we examined differentially expressed genes (DEGs) in mouse MCs following CGRP treatment. The expression of key genes in colonic MCs and their relationship with CGRP-containing fibers were examined by immunofluorescence in chronic water-avoidance stress (WAS)-induced visceral hyperalgesia mice. A total of 29 DEGs were found significantly changed with 28 upregulated and 1 downregulated following treatment of MCs with CGRP. Bioinformatics analysis showed that key higher DEGs included those associated with response to corticotropin-releasing hormone (CRH), regulation of transcription, MC activation, and proliferation. These processes are enriched for genes associated with stress-induced MC activation in IBS. Western blot verified changes in representative DEGs (Nr4a3, Crem, Gpr35, FosB, Sphlk1) and real-time cell analysis (RTCA) verified the MC proliferation. The vast majority of colonic MCs nearly CGRP-containing fibers in WAS mice overexpressed only Nr4a3 with little to no FosB, Gpr35, Sphlk1, or Crem expression. Nr4a3 knockdown may attenuate the promotion effect of CGRP on MC viability. Our results suggest that CGRP is a critical regulator of key expressed genes in MC activation. Nr4a3 as a novel regulator of MC function may have an effect on stress-induced visceral hyperalgesia, and this may represent the novel target for drug development.

Histone H3K9 methylation regulates chronic stress and IL-6-induced colon epithelial permeability and visceral pain.

Chronic stress is associated with activation of the HPA axis, elevation in pro-inflammatory cytokines, decrease in intestinal epithelial cell tight junction (TJ) proteins, and enhanced visceral pain. It is unknown whether epigenetic regulatory pathways play a role in chronic stress-induced intestinal barrier dysfunction and visceral hyperalgesia. Young adult male rats were subjected to water avoidance stress±H3K9 methylation inhibitors or siRNAs. Visceral pain response was assessed. Differentiated Caco-2/BBE cells and human colonoids were treated with cortisol or IL-6±antagonists. Expression of TJ, IL-6, and H3K9 methylation status at gene promoters was measured. Transepithelial electrical resistance and FITC-dextran permeability were evaluated. Chronic stress induced IL-6 up-regulation prior to a decrease in TJ proteins in the rat colon. The IL-6 level inversely correlated with occludin expression. Treatment with IL-6 decreased occludin and induced visceral hyperalgesia. Chronic stress and IL-6 increased H3K9 methylation and decreased transcriptional GR binding to the occludin gene promoter, leading to down-regulation of protein expression and increase in paracellular permeability. Intrarectal administration of a H3K9 methylation antagonist prevented chronic stress-induced visceral hyperalgesia in the rat. In a human colonoid model, cortisol decreased occludin expression, which was prevented by the GR antagonist RU486, and IL-6 increased H3K9 methylation and decreased TJ protein levels, which were prevented by inhibitors of H3K9 methylation. Our findings support a novel role for methylation of the repressive histone H3K9 to regulate chronic stress, pro-inflammatory cytokine-mediated reduction in colon TJ protein levels, and increase in paracellular permeability and visceral hyperalgesia.

Down-regulation of HTR1A-modulated ACC activation contributes to stress-induced visceral hyperalgesia in rats.

Long-term stress was suggested to cause visceral hypersensitivity and promote functional gastrointestinal disorders (FGIDs). Some brain regions such as the anterior cingulate cortex (ACC) may play an important role for generating visceral hypersensitivity; however, its molecular mechanisms are not clear. This study aimed to explore the role of 5-HT1A receptors (HTR1As) in activating ACC and corresponding mechanism, in stress-induced visceral hyperalgesia rats. The VH rat model was established by chronic water avoidance stress (WAS), and the visceral sensitivity was measured by electromyogram. Rat's anxiety-like behaviors were evaluated by the open field test (OFT) and elevated plus maze (EPM). To overexpress or down-regulate HTR1A expression, HTR1A-specific lentivirus expressing the green fluorescent protein was administered into the ACC. Protein expression levels were observed by Western blot. The protein expression of HTR1A in bilateral ACC in WAS group was significantly lower than that in normal control (NC) and Sham-WAS groups, while the levels of c-fos in the ACC of WAS rats were significantly higher. Down-regulation of HTR1As could induce VH in control rats with the increased expression of c-fos, p-ERK, and p-Akt in ACC, while up-regulation of HTR1As in the ACC could partly inhibit ACC sensitization and stress-induced visceral hyperalgesia. Down-regulation of HTR1As modulates ACC activation probably through activating ERK and Akt pathways, thus contributes to the formation of stress-induced visceral hyperalgesia.

Cortisol affects pain sensitivity and pain-related emotional learning in experimental visceral but not somatic pain: a randomized controlled study in healthy men and women.

Despite growing interest in the role of stress mediators in pain chronicity, the effects of the stress hormone cortisol on acute pain remain incompletely understood. In a randomized, double-blind, placebo-controlled study with N = 100 healthy volunteers, we tested the effects of oral hydrocortisone (20 mg) in 2 widely used pain models for the visceral and somatic modality. Salivary cortisol was increased in the hydrocortisone group (time × group: P < 0.001). For the visceral modality, assessed using pressure-controlled rectal distensions, hydrocortisone decreased the pain threshold from before to after treatment (time × group: P = 0.011), an effect primarily driven by women (time × sex: P = 0.027). For the somatic modality, cutaneous heat pain thresholds remained unaffected by hydrocortisone. Hydrocortisone did not alter perceived pain intensity or unpleasantness of either modality. Conditioned pain-related fear in response to predictive cues was only observed for the visceral modality (time × modality: P = 0.026), an effect that was significantly reduced by hydrocortisone compared with placebo (time × group: P = 0.028). This is the first psychopharmacological study to support that acutely increased cortisol enhances pain sensitivity and impairs pain-related emotional learning within the visceral, but not the somatic pain modality. Stress-induced visceral hyperalgesia and deficits in emotional pain-related learning could play a role in the pathophysiology of chronic visceral pain.

Chronic stress and intestinal permeability: Lubiprostone regulates glucocorticoid receptor-mediated changes in colon epithelial tight junction proteins, barrier function, and visceral pain in the rodent and human.

Chronic psychological stress is associated with increased intestinal epithelial permeability and visceral hyperalgesia. Lubiprostone, an agonist for chloride channel-2, promotes secretion and accelerates restoration of injury-induced epithelial barrier dysfunction. The mechanisms underlying how lubiprostone regulates colon epithelial barrier function and visceral hyperalgesia in chronic stress remain unknown. Male rats were subjected to water avoidance stress for 10 consecutive days. Lubiprostone was administered daily during the stress phase. Visceromotor response to colorectal distension was measured. Human colon crypts and cell lines were treated with cortisol and lubiprostone. The transepithelial electrical resistance and FITC-dextran permeability were assayed. Chromatin immunoprecipitation was conducted to assess glucocorticoid receptor binding at tight junction gene promoters. Lubiprostone significantly decreased chronic stress-induced visceral hyperalgesia in the rat (P<0.05; n=6). WA stress decreased occludin and claudin-1 and increased claudin-2 in rat colon crypts, which was prevented by lubiprostone. Cortisol treatment induced similar alterations of tight junction protein expression in Caco-2/BBE cells (P<0.05) and significantly changed paracellular permeability in monolayers (P<0.01). These changes were blocked by lubiprostone. Glucocorticoid receptor and its binding at occludin promoter region were decreased in cortisol-treated cells and human colon crypts, which was largely reversed by lubiprostone. In rat colonic cells, glucocorticoid receptor and its co-chaperone proteins were down-regulated after corticosterone treatment and lubiprostone reversed these changes. Lubiprostone preferentially prevents chronic stress-induced alterations of intestinal epithelial tight junctions, barrier function, and visceral hyperalgesia that was associated with modulation of glucocorticoid receptor expression and function.

Su1945 Histone Deacetylases Regulate Epithelial Tight Junction Protein Expression and Prevent Chronic Stress-induced Visceral Hyperalgesia

Su1945 Histone Deacetylases Regulate Epithelial Tight Junction Protein Expression and Prevent Chronic Stress-induced Visceral Hyperalgesia

Chronic stress and peripheral pain: Evidence for distinct, region-specific changes in visceral and somatosensory pain regulatory pathways

Chronic stress alters the hypothalamic–pituitary–adrenal (HPA) axis and enhances visceral and somatosensory pain perception. It is unresolved whether chronic stress has distinct effects on visceral and somatosensory pain regulatory pathways. Previous studies reported that stress-induced visceral hyperalgesia is associated with reciprocal alterations of endovanilloid and endocannabinoid pain pathways in DRG neurons innervating the pelvic viscera. In this study, we compared somatosensory and visceral hyperalgesia with respect to differential responses of peripheral pain regulatory pathways in a rat model of chronic, intermittent stress. We found that chronic stress induced reciprocal changes in the endocannabinoid 2-AG (increased) and endocannabinoid degradation enzymes COX-2 and FAAH (decreased), associated with down-regulation of CB1 and up-regulation of TRPV1 receptors in L6–S2 DRG but not L4–L5 DRG neurons. In contrast, sodium channels Nav1.7 and Nav1.8 were up-regulated in L4–L5 but not L6–S2 DRGs in stressed rats, which was reproduced in control DRGs treated with corticosterone in vitro. The reciprocal changes of CB1, TRPV1 and sodium channels were cell-specific and observed in the sub-population of nociceptive neurons. Behavioral assessment showed that visceral hyperalgesia persisted, whereas somatosensory hyperalgesia and enhanced expression of Nav1.7 and Nav1.8 sodium channels in L4–L5 DRGs normalized 3days after completion of the stress phase. These data indicate that chronic stress induces visceral and somatosensory hyperalgesia that involves differential changes in endovanilloid and endocannabinoid pathways, and sodium channels in DRGs innervating the pelvic viscera and lower extremities. These results suggest that chronic stress-induced visceral and lower extremity somatosensory hyperalgesia can be treated selectively at different levels of the spinal cord.

Identification of Spinal Cord MicroRNA and Gene Signatures in a Model of Chronic Stress-Induced Visceral Hyperalgesia in Rat.

IntroductionAnimal studies have shown that stress could induce epigenetic and transcriptomic alterations essential in determining the balance between adaptive or maladaptive responses to stress. We tested the hypothesis that chronic stress in rats deregulates coding and non-coding gene expression in the spinal cord, which may underline neuroinflammation and nociceptive changes previously observed in this model.MethodsMale Wistar rats were exposed to daily stress or handled, for 10 days. At day 11, lumbar spinal segments were collected and processed for mRNA/miRNA isolation followed by expression profiling using Agilent SurePrint Rat Exon and Rat miRNA Microarray platforms. Differentially expressed gene lists were generated using the dChip program. Microarrays were analyzed using the Ingenuity Pathways Analysis (IPA) tool from Ingenuity Systems. Multiple methods were used for the analysis of miRNA-mRNA functional modules. Quantitative real time RT-PCR for Interleukin 6 signal transducer (gp130), the Signal Transducer And Activator Of Transcription 3 (STAT3), glial fibrillary acidic protein and mir-17-5p were performed to confirm levels of expression.ResultsGene network analysis revealed that stress deregulated different inflammatory (IL-6, JAK/STAT, TNF) and metabolic (PI3K/AKT) signaling pathways. MicroRNA array analysis revealed a signature of 39 deregulated microRNAs in stressed rats. MicroRNA-gene network analysis showed that microRNAs are regulators of two gene networks relevant to inflammatory processes. Specifically, our analysis of miRNA-mRNA functional modules identified miR-17-5p as an important regulator in our model. We verified miR-17-5p increased expression in stress using qPCR and in situ hybridization. In addition, we observed changes in the expression of gp130 and STAT3 (involved in intracellular signaling cascades in response to gp130 activation), both predicted targets for miR-17-5p. A modulatory role of spinal mir17-5p in the modulation of visceral sensitivity was confirmed in vivo.ConclusionUsing an integrative high throughput approach, our findings suggest a link between miR-17-5p increased expression and gp130/STAT3 activation providing new insight into the possible mechanisms mediating the effect of chronic stress on neuroinflammation in the spinal cord.

Tu1876 Chronic Stress-Induced Visceral Hyperalgesia: Evidence for Differential Effects of Lubiprostone on Tight Junction Protein Expression in the Colon

Introduction: In humans, chronic psychological stress is associated with increased intestinal permeability and visceral hyperalgesia, which is recapitulated in the chronic intermittent water avoidance stress (WAS) rat model. Previously, we reported that WAS rats demonstrated increased intestinal permeability for macromolecules specifically in the colon, which correlated with reduced epithelial tight junction protein expression in the colon epithelium. Lubiprostone (Lub), a prostaglandin receptor agonist used to treat chronic constipation and IBS-C, has been shown to restore barrier function and epithelial tight junction protein expression in the ischemic porcine intestine. Hypothesis: Lub treatment ameliorates increased colon permeability and visceral hyperalgesia in the WAS rat model. Methods: Male Sprague Dawley rats were subjected to one hour of WAS for 10 consecutive days with or without oral administration of Lub. A 20 μg/kg/day dose of Lub was chosen based on a dose response study. On day 11, visceral motor response (VMR) to colorectal distention (CRD) or colon permeability were measured. Permeability was assayed by measuring plasma levels of the non-ionic molecule FITC-Dextran (4KDa), which was given by gavage at 400 mg/Kg. Distal colon tissue was used for Western blot analysis. Results: WAS was associated with increased colon permeability compared to controls in 7 of 14 rats (p = 0.0036) and a decrease in the level of the epithelial tight junction protein occludin (p < 0.05). Lub prevented the stress-induced increase in colon permeability (p = 0.01), as well as the decrease in occludin. WAS rats had a significant increase in pellet output over controls (p < 0.0001), however there were no significant difference in pellet output between Lub treated and untreated WAS rats. All WAS rats showed visceral hyperalgesia in response to CRD (60 mmHg, p < 0.0001) in comparison to controls. Lub treatment of WAS rats prevented visceral hyperalgesia in response to CRD for 7 out of 14 rats (p < 0.0001), whereas the other 7 had VMR to CRD levels equivalent to untreated WAS rats. Conclusion: Lub prevents WASassociated: 1) decrease in epithelial tight junction protein expression in a subset of rats that showed an increase in colon permeability and 2) hyperalgesia in a subset of rats, despite genetic homogeneity. We hypothesize that increased gut permeability and hyperalgesia in response to WAS are linked, and the differential effects of WAS on colon permeability and Lub treatment on visceral hyperalgesia reflects differences in epigenetic regulation of relevant target genes.

60 Chronic Stress-Induced Visceral Hyperalgesia: Evidence for Glucocorticoid Receptor (GR)-Mediated Down-Regulation of Claudin-1 Transcription in the Rat Colon

60 Chronic Stress-Induced Visceral Hyperalgesia: Evidence for Glucocorticoid Receptor (GR)-Mediated Down-Regulation of Claudin-1 Transcription in the Rat Colon

742 Chronic Stress-Induced Visceral Hyperalgesia: Molecular Evidence for Corticosterone (CORT)-Mediated Reduction in Cannabinoid Receptor Type 1 (CB1) via Functional Glucocorticoid Response Element (Gre) on CB1 Promoter in an Epigenetic Manner

742 Chronic Stress-Induced Visceral Hyperalgesia: Molecular Evidence for Corticosterone (CORT)-Mediated Reduction in Cannabinoid Receptor Type 1 (CB1) via Functional Glucocorticoid Response Element (Gre) on CB1 Promoter in an Epigenetic Manner

Alterations in Prefrontal-Limbic Functional Activation and Connectivity in Chronic Stress-Induced Visceral Hyperalgesia

Repeated water avoidance stress (WAS) induces sustained visceral hyperalgesia (VH) in rats measured as enhanced visceromotor response to colorectal distension (CRD). This model incorporates two characteristic features of human irritable bowel syndrome (IBS), VH and a prominent role of stress in the onset and exacerbation of IBS symptoms. Little is known regarding central mechanisms underlying the stress-induced VH. Here, we applied an autoradiographic perfusion method to map regional and network-level neural correlates of VH. Adult male rats were exposed to WAS or sham treatment for 1 hour/day for 10 days. The visceromotor response was measured before and after the treatment. Cerebral blood flow (CBF) mapping was performed by intravenous injection of radiotracer ([14C]-iodoantipyrine) while the rat was receiving a 60-mmHg CRD or no distension. Regional CBF-related tissue radioactivity was quantified in autoradiographic images of brain slices and analyzed in 3-dimensionally reconstructed brains with statistical parametric mapping. Compared to sham rats, stressed rats showed VH in association with greater CRD-evoked activation in the insular cortex, amygdala, and hypothalamus, but reduced activation in the prelimbic area (PrL) of prefrontal cortex. We constrained results of seed correlation analysis by known structural connectivity of the PrL to generate structurally linked functional connectivity (SLFC) of the PrL. Dramatic differences in the SLFC of PrL were noted between stressed and sham rats under distension. In particular, sham rats showed negative correlation between the PrL and amygdala, which was absent in stressed rats. The altered pattern of functional brain activation is in general agreement with that observed in IBS patients in human brain imaging studies, providing further support for the face and construct validity of the WAS model for IBS. The absence of prefrontal cortex-amygdala anticorrelation in stressed rats is consistent with the notion that impaired corticolimbic modulation acts as a central mechanism underlying stress-induced VH.

898 Chronic Stress-Induced Visceral Hyperalgesia: Evidence That the Formation of CB1 and TRPV1 Receptor Complexes Modulates Pain in Dorsal Root Ganglion (DRG) Neurons

Introduction: PGD2 is an important mediator released after mast cell activation. Our previous studies demonstrated PGD2 sensitizes esophageal nodose C-fibers to increase their response to esophageal distension. This study aims to determine the underlying mechanism of the sensitization process. Methods: PGD2 DP1 receptor expressions in nodose neurons were determined by immunofluorescent-staining, Western blot, and RT-PCR. Extracellular single fiber recordings were performed from vagal nodose neurons using ex vivo esophageal-vagal preparations with intact nerve endings in the esophagus. We recorded action potentials evoked by esophageal distensions before and after perfusion of PGD2 with/without pretreatment of DP1 receptor antagonist BWA 868C. Whole-cell patch-clamp recording was performed to investigate the action of PGD2 on isolated nodose neurons. Viscermotor responses (VMR) to esophageal distension were recorded by electromyography from acromiotrapezius muscles in animals receiving PGD2 injection (20 μl, 100 nM) into the wall of the esophagus before and after truncal vagatomy. Results: DP1 receptor expressions were identified in nodose neurons by immunofluorescent-labeling, Western blot, and RT-PCR. 98% of DP1positive neurons were of smalland medium-diameters (φ 0.05, n=4). Whole-cell patch-clamp recording from isolated nodose neurons demonstrated PGD2 (1 μM) depolarized recorded neurons (13±2 mV, n=5) accompanied by an increase in the neuronal input resistance from 360±20 to 420±25 MΩ. IV relationship studies showed current reversed at 105 mV suggesting the effect was mediated by the closure of K+ channel(s). Compared to controls, PGD2 injection into the esophagus significantly enhanced esophageal distension evoked-VMRs at distension pressures 20, 40, 60, and 80 mmHg respectively (p <0.05, n=3). Left truncal vagatomy attenuated PGD2induced enhancements of VMR in response to esophageal distension (p <0.05, n=3) indicating the participation of vagal afferent pathway in this sensitization process. Conclusion: These data demonstrate mast cell mediator PGD2 plays a crucial role in sensitization of esophageal nodose C-fiber neurons to induce esophageal mechanical hypersensitivity. This sensitization effect is mediated via DP1 receptor and involves downstream K+ channel.

Early-life stress induces visceral hypersensitivity in mice

Early-life stress is a risk factor for irritable bowel syndrome (IBS), a common and debilitating functional gastrointestinal disorder that is often co-morbid with stress-related psychiatric disorders. In the rat, maternal separation (MS) stress has been shown to induce visceral hypersensitivity in adulthood and thus has become a useful model of IBS. However, development of mouse models of maternal separation has been difficult. Given the advent of transgenic mouse technology, such models would be useful to further our understanding of the pathophysiology of IBS and to develop new pharmacological treatments. Thus, the present study aimed to develop a mouse model of MS stress-induced visceral hyperalgesia as measured using manometric recordings of colorectal distension (CRD). Moreover, since the GABAB receptor has been reported to play a role in pain processes, we also assessed its role in visceral nociception using novel GABAB(1b) receptor subunit knockout mice.CRD was performed in adult male wildtype and GABAB(1b) receptor knockout mice that had undergone unpredictable MS combined with unpredictable maternal stress (MSUS) from postnatal day 1 through 14 (PND 1–14). MSUS induced visceral hypersensitivity in both wildtype and GABAB(1b) receptor knockout mice when compared with non-stressed mice. Wildtype and GABAB(1b) receptor knockout mice did not differ in baseline or stress-induced visceral sensitivity. To the best of our knowledge, this is the first study to show that early-life stress induces visceral hypersensitivity in a mouse model. These findings may provide a novel mouse model of visceral hypersensitivity which may aid our understanding of its underlying mechanisms in future studies.

Role of astrocytes and altered regulation of spinal glutamatergic neurotransmission in stress-induced visceral hyperalgesia in rats.

Glutamate (Glu) is the primary excitatory neurotransmitter in the central nervous system and plays a critical role in the neuroplasticity of nociceptive networks. We aimed to examine the role of spinal astroglia in the modulation of glutamatergic neurotransmission in a model of chronic psychological stress-induced visceral hyperalgesia in male Wistar rats. We assessed the effect of chronic stress on different glial Glu control mechanisms in the spinal cord including N-methyl-d-aspartate receptors (NMDARs), glial Glu transporters (GLT1 and GLAST), the Glu conversion enzyme glutamine synthetase (GS), and glial fibrillary acidic protein (GFAP). We also tested the effect of pharmacological inhibition of NMDAR activation, of extracellular Glu reuptake, and of astrocyte function on visceral nociceptive response in naive and stressed rats. We observed stress-induced decreased expression of spinal GLT1, GFAP, and GS, whereas GLAST expression was upregulated. Although visceral hyperalgesia was blocked by pharmacological inhibition of spinal NMDARs, we observed no stress effects on NMDAR subunit expression or phosphorylation. The glial modulating agent propentofylline blocked stress-induced visceral hyperalgesia, and blockade of GLT1 function in control rats resulted in enhanced visceral nociceptive response. These findings provide evidence for stress-induced modulation of glia-controlled spinal Glu-ergic neurotransmission and its involvement in chronic stress-induced visceral hyperalgesia. The findings reported in this study demonstrate a unique pattern of stress-induced changes in spinal Glu signaling and metabolism associated with enhanced responses to visceral distension.

Cytokine Modulation of Visceral Afferents via Cation Channels is Switched in Chronic Visceral Hypersensitivity

Introduction: Increasing evidence suggests that chronic stress alters behavior and modifies epigenetic regulation of genes in the central nervous system. DNA methylation, catalyzed by DNA methyltransferases (DNMTs), is an important epigenetic mechanism of transcriptional control of gene expression. We reported previously that chronic psychological stress induced visceral hyperalgesia and differential alterations in the expression of several genes in DRG neurons innervating the colon in the rat. A potential role for epigenetic regulation in peripheral sensory pathways has not been investigated. Objectives: We examined the hypothesis that DNMTs play an important role in the regulation of chronic stress-induced visceral hyperalgesia. Methods: Male rats were exposed to 1-hour water avoidance (WA) stress daily for 10 consecutive days as a chronic stress paradigm. SiRNA for DNMT1 was administrated in situ to L6-S2 DRGs every other day during the stress procedure. The visceromotor response (VMR) to colorectal distension was measured. Retrograde labeling with cholera toxin B (CTB)-FITCwas employed to identify colonDRG neurons. Immunofluorescence andWestern blot analysis were used to assess protein expression. In Vitro studies were performed in isolated control DRGs in the presence or absence of corticosterone (CORT; 10 μM) and RU-486 (corticoid receptor antagonist, 500 nM). Results: WA stress rats demonstrated significant increases in the level of DNMT1 and DNMT3b but not DNMT3a in L6-S2 DRGs compared with the controls. Enzyme activity assessment showed a 42% increase in DNMT1 activity in L6-S2 DRGs in stressed rats. Immunofluorescence studies revealed a significant increase in DNMT1 in small-sized, C-fiber neurons in WA stressed rats (52.3±2.2%) compared with the control (31.7±1.6%). Retrograde labeling demonstrated that 72.0±2.1% of the CTB-FITC labeled colonic DRG neurons were positive for DNMT1 in stressed rats compared to 40.0±6.5% in controls (P<0.05; n=4). The VMR in WA stressed rats was increased 68% and 92% above control responses at pressures of 40 and 60 mm Hg, respectively. Treatment of stressed rats with siRNA for DNMT1 prevented the VMR enhancement and changes in DNMT1 proteins levels in L6-S2 DRGs. In addition, treatment of control L6-S2 DRGs In Vitro with CORT (10 μM) increased DNMT1 expression level that was prevented by RU-486 (500 nM) (P<0.05). Conclusions: These data support the novel and provocative interpretation that: 1. Chronic stress induces epigenetic regulation of genes in primary nociceptive neurons; 2. DNA methyltransferase 1 (DNMT1) plays an important role in modulation of chronic stress-induced visceral hyperalgesia; and 3. DNA methyltransferases represent a potential target for treatment of functional GI disorders associated with visceral hyperalgesia.

Identification of Colonic Afferent Central Terminals and Changes Following Colonic Inflammation

Introduction: Increasing evidence suggests that chronic stress alters behavior and modifies epigenetic regulation of genes in the central nervous system. DNA methylation, catalyzed by DNA methyltransferases (DNMTs), is an important epigenetic mechanism of transcriptional control of gene expression. We reported previously that chronic psychological stress induced visceral hyperalgesia and differential alterations in the expression of several genes in DRG neurons innervating the colon in the rat. A potential role for epigenetic regulation in peripheral sensory pathways has not been investigated. Objectives: We examined the hypothesis that DNMTs play an important role in the regulation of chronic stress-induced visceral hyperalgesia. Methods: Male rats were exposed to 1-hour water avoidance (WA) stress daily for 10 consecutive days as a chronic stress paradigm. SiRNA for DNMT1 was administrated in situ to L6-S2 DRGs every other day during the stress procedure. The visceromotor response (VMR) to colorectal distension was measured. Retrograde labeling with cholera toxin B (CTB)-FITCwas employed to identify colonDRG neurons. Immunofluorescence andWestern blot analysis were used to assess protein expression. In Vitro studies were performed in isolated control DRGs in the presence or absence of corticosterone (CORT; 10 μM) and RU-486 (corticoid receptor antagonist, 500 nM). Results: WA stress rats demonstrated significant increases in the level of DNMT1 and DNMT3b but not DNMT3a in L6-S2 DRGs compared with the controls. Enzyme activity assessment showed a 42% increase in DNMT1 activity in L6-S2 DRGs in stressed rats. Immunofluorescence studies revealed a significant increase in DNMT1 in small-sized, C-fiber neurons in WA stressed rats (52.3±2.2%) compared with the control (31.7±1.6%). Retrograde labeling demonstrated that 72.0±2.1% of the CTB-FITC labeled colonic DRG neurons were positive for DNMT1 in stressed rats compared to 40.0±6.5% in controls (P<0.05; n=4). The VMR in WA stressed rats was increased 68% and 92% above control responses at pressures of 40 and 60 mm Hg, respectively. Treatment of stressed rats with siRNA for DNMT1 prevented the VMR enhancement and changes in DNMT1 proteins levels in L6-S2 DRGs. In addition, treatment of control L6-S2 DRGs In Vitro with CORT (10 μM) increased DNMT1 expression level that was prevented by RU-486 (500 nM) (P<0.05). Conclusions: These data support the novel and provocative interpretation that: 1. Chronic stress induces epigenetic regulation of genes in primary nociceptive neurons; 2. DNA methyltransferase 1 (DNMT1) plays an important role in modulation of chronic stress-induced visceral hyperalgesia; and 3. DNA methyltransferases represent a potential target for treatment of functional GI disorders associated with visceral hyperalgesia.

Chronic Stress-Induced Visceral Hyperalgesia: Evidence for Epigenetic Regulation of DNA Methyltransferase I (Dnmt1) in Dorsal Root Ganglion (DRG) Neurons Innervating the Colon in the Rat

Introduction: Increasing evidence suggests that chronic stress alters behavior and modifies epigenetic regulation of genes in the central nervous system. DNA methylation, catalyzed by DNA methyltransferases (DNMTs), is an important epigenetic mechanism of transcriptional control of gene expression. We reported previously that chronic psychological stress induced visceral hyperalgesia and differential alterations in the expression of several genes in DRG neurons innervating the colon in the rat. A potential role for epigenetic regulation in peripheral sensory pathways has not been investigated. Objectives: We examined the hypothesis that DNMTs play an important role in the regulation of chronic stress-induced visceral hyperalgesia. Methods: Male rats were exposed to 1-hour water avoidance (WA) stress daily for 10 consecutive days as a chronic stress paradigm. SiRNA for DNMT1 was administrated in situ to L6-S2 DRGs every other day during the stress procedure. The visceromotor response (VMR) to colorectal distension was measured. Retrograde labeling with cholera toxin B (CTB)-FITCwas employed to identify colonDRG neurons. Immunofluorescence andWestern blot analysis were used to assess protein expression. In Vitro studies were performed in isolated control DRGs in the presence or absence of corticosterone (CORT; 10 μM) and RU-486 (corticoid receptor antagonist, 500 nM). Results: WA stress rats demonstrated significant increases in the level of DNMT1 and DNMT3b but not DNMT3a in L6-S2 DRGs compared with the controls. Enzyme activity assessment showed a 42% increase in DNMT1 activity in L6-S2 DRGs in stressed rats. Immunofluorescence studies revealed a significant increase in DNMT1 in small-sized, C-fiber neurons in WA stressed rats (52.3±2.2%) compared with the control (31.7±1.6%). Retrograde labeling demonstrated that 72.0±2.1% of the CTB-FITC labeled colonic DRG neurons were positive for DNMT1 in stressed rats compared to 40.0±6.5% in controls (P<0.05; n=4). The VMR in WA stressed rats was increased 68% and 92% above control responses at pressures of 40 and 60 mm Hg, respectively. Treatment of stressed rats with siRNA for DNMT1 prevented the VMR enhancement and changes in DNMT1 proteins levels in L6-S2 DRGs. In addition, treatment of control L6-S2 DRGs In Vitro with CORT (10 μM) increased DNMT1 expression level that was prevented by RU-486 (500 nM) (P<0.05). Conclusions: These data support the novel and provocative interpretation that: 1. Chronic stress induces epigenetic regulation of genes in primary nociceptive neurons; 2. DNA methyltransferase 1 (DNMT1) plays an important role in modulation of chronic stress-induced visceral hyperalgesia; and 3. DNA methyltransferases represent a potential target for treatment of functional GI disorders associated with visceral hyperalgesia.

Chronic Stress-Induced Visceral Hyperalgesia: Evidence for Region-Specific, Corticosterone (CRT)-Mediated Reduction in Colon Epithelial Tight Junction Protein Levels and Increased Permeability to Macromolecules

G A A b st ra ct s 10d, EAAT2 activator) normalized the VMR to CRD in VH rats. On the other hand, microinfusion of ACC with dihydrokainate (20 mM for 10 d, EAAT2 blocker) in normal rats resulted in enhanced VMR responses similar to those observed in VH rats. In conclusion, we showed that downregulation of EAAT2 in ACC occurs in the VH state. This appears to be negatively regulated by the ET system. Delayed clearance of extracellular glutamate as a result of reduced expression of EAAT2 plays a critical role in the pathogenesis of visceral hypersensitivity. Agents that modulate glutamate reuptake system may provide new therapeutic options to the treatment of visceral hypersensitivity disorders.