Chronic Visceral Hyperalgesia Research Articles

Retracted: NUCB2/Nesfatin-1 Regulation of Chronic Visceral Hyperalgesia

Retracted: NUCB2/Nesfatin-1 Regulation of Chronic Visceral Hyperalgesia

NGF/PI3K/TRPV1 pathway mediates the regulation of visceral pain by acupuncture at "Shangjuxu" (ST37) in irritable bowel syndrome rats with chronic visceral hyperalgesia.

To investigate the effect of manual acupuncture at "Shangjuxu"(ST37) on nerve growth factor(NGF)/phosphatidylinositol 3-kinase(PI3K)/transient receptor potential vanilloid 1(TRPV1) signaling pathway in rats with chronic visceral hyperalgesia of irritable bowel syndrome (IBS), so as to explore its underlying mechanism in treating IBS chronic visceral hyperalgesia. IBS chronic visceral hyperalgesia model was established by colorectal dilation stimulation for 2 weeks for SD pups at 8 d after birth, which were fed until 8-week age after the stimulation. Then the verified successfully modeled adult rats were randomly divided into model, Shangjuxu, and non-acupoint groups, with 6 rats in each group, and 6 unmodeled rats were selected as normal group. On the next day of model evaluation, rats in the Shangjuxu group received acupuncture at right ST37 while rats in the non-acupoint group received acupuncture at the non-meridian and non-acupoint point in the right hypochondrium, both for 15 min, with manual twisting of mild reinforcing and reducing performed for 30 s at an interval of 5 min, once a day, for a total of 7 d. Abdominal withdrawal reflex(AWR) score was used to evaluate the degree of chronic visceral pain in rats. Western blot and real-time fluorescence quantitative PCR were used to detect the colonic protein and mRNA expressions of NGF, tropomyosin receptor kinase A (TrkA), PI3K and TRPV1. The positive expressions of PI3K and TRPV1 proteins in the colon of rats were detected by immunohistochemistry method. Compared with the normal group, AWR scores corresponding to 4 pressure levels of 20, 40, 60 and 80 mm Hg, mRNA and protein expressions of NGF, TrkA, PI3K and TRPV1 in colon tissue, and positive expressions of PI3K and TRPV1 in colon tissue were significantly increased(P<0.05) in the model group. After intervention, compared with the model group, rats in the Shangjuxu group had reduced AWR scores corresponding to 4 pressure levels of 20, 40, 60 and 80 mm Hg, lower colonic mRNA and protein expressions of NGF, TrkA, PI3K and TRPV1, and decreased positive expressions of PI3K and TRPV1 in colon tissue(P<0.05), while there were no significant differences in the above indexes of the non-acupoint group. Manual acupuncture at ST37 can alleviate IBS chronic visceral hyperalgesia in rat and its analgesic effect may be related to regulating NGF/PI3K/TRPV1 signaling pathway.

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.

NUCB2/Nesfatin-1 Regulation of Chronic Visceral Hyperalgesia.

Objective We previously described that different concentration Nucleobindin-2 (NUCB2)/Nesfatin-1 gradients differently regulated visceral hypersensitivity in irritable bowel syndrome. Therefore, this study is aimed at evaluating the effect of NUCB2/Nesfatin-1 on model rats with chronic visceral hyperalgesia. Methods Neonatal and mature Sprague-Dawley rats were randomly divided into the healthy control and chronic visceral hyperalgesia model groups. The model was built by combining maternal separation with the acetic acid enema. The models were identified by the distension volume threshold to reach abdominal withdraw reflex (AWR) score = 3, histological staining, and myeloperoxidase (MPO) detection. The visceral sensitivity to chronic visceral hyperalgesia was then evaluated. Result Rats in the model group responded more strongly to pulling stimulation than healthy controls; the distension volume threshold causing AWR3 response in model rats was lower than the control group before NUCB2/Nesfatin-1 intervention. After intervention, the distension volume threshold was significantly lower in the NUCB2/Nesfatin-1 central intervention group than in the NUCB2/Nesfatin-1 peripheral intervention group, and the peak value of external oblique muscle electrical activity was significantly higher. Additionally, compared with the male intervention group, in the female intervention group, the volume threshold was significantly lower and the peak value was higher. Conclusion NUCB2/Nesfatin-1 could regulate visceral sensitivity in chronic visceral hyperalgesia model rats; its regulatory effect correlated with the type of NUCB2/Nesfatin-1 intervention approaches (central or peripheral) and sex (male or female).

Microglia-derived TNF-α inhibiting GABAergic neurons in the anterior lateral bed nucleus of the stria terminalis precipitates visceral hypersensitivity induced by colorectal distension in rats

Irritable bowel syndrome (IBS) is a common and debilitating gastrointestinal disorder that is exacerbated by stress and characterized by abdominal pain. Although microglia in the CNS have been implicated as an important mediator of the stress response, the role of microglia and microglia-GABAergic neuron interactions in the limbic area, most notably BNST, in the development of colorectal hypersensitivity has not been determined. We established a neonatal colorectal distension-induced chronic visceral hyperalgesia model in rats. The results showed that the frequency of spontaneous discharges of alBNST GABAergic neurons and the expression of GAD65/67 were significantly decreased in rats with chronic visceral pain. Moreover, ablation of BNST GABAergic neurons significantly reduced the visceral pain threshold in normal rats. Meanwhile, the number of M1 proinflammatory microglia and the expression of the M1 proinflammatory microglia-derived cytokines IL-6 and TNF-α were increased in the alBNST of rats with chronic visceral pain. Furthermore, alBNST infusion of the microglial inhibitor minocycline or IL-6 and TNF-α neutralizing antibodies significantly increased the visceral pain threshold. The decreased frequency of spontaneous discharges of alBNST GABAergic neurons in rats with chronic visceral pain was mimicked by a bath perfusion of TNF-α, but not IL-6, and was abolished by a perfusion of the microglial inhibitor minocycline. In addition, the alBNST infusion of the microglial inhibitor minocycline upregulated the expression of GAD65/67. Moreover, ablation of BNST GABAergic neurons significantly decreased the visceral pain threshold in normal rats, which was not reversed by a subsequent infusion of the microglial inhibitor minocycline. Our findings revealed this microglia-GABAergic neuron circuit in the alBNST, and this microglia-driven disinhibitory mechanism is essential for brain and gut dysfunction in stressful condition, providing a novel potential target for treating patients with IBS presenting visceral pain that is worsened during episodes of stress.

The Role of Epigenomic Regulatory Pathways in the Gut-Brain Axis and Visceral Hyperalgesia.

The gut-brain axis (GBA) is broadly accepted to describe the bidirectional circuit that links the gastrointestinal tract with the central nervous system (CNS). Interest in the GBA has grown dramatically over past two decades along with advances in our understanding of the importance of the axis in the pathophysiology of numerous common clinical disorders including mood disorders, neurodegenerative disease, diabetes mellitus, non-alcohol fatty liver disease (NAFLD) and enhanced abdominal pain (visceral hyperalgesia). Paralleling the growing interest in the GBA, there have been seminal developments in our understanding of how environmental factors such as psychological stress and other extrinsic factors alter gene expression, primarily via epigenomic regulatory mechanisms. This process has been driven by advances in next-generation multi-omics methods and bioinformatics. Recent reviews address various components of GBA, but the role of epigenomic regulatory pathways in chronic stress-associated visceral hyperalgesia in relevant regions of the GBA including the amygdala, spinal cord, primary afferent (nociceptive) neurons, and the intestinal barrier has not been addressed. Rapidly developing evidence suggests that intestinal epithelial barrier dysfunction and microbial dysbiosis play a potentially significant role in chronic stress-associated visceral hyperalgesia in nociceptive neurons innervating the lower intestine via downregulation in intestinal epithelial cell tight junction protein expression and increase in paracellular permeability. These observations support an important role for the regulatory epigenome in the development of future diagnostics and therapeutic interventions in clinical disorders affecting the GBA.

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.

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.

Chronic stress-associated visceral hyperalgesia correlates with severity of intestinal barrier dysfunction.

In humans, chronic psychological stress is associated with increased intestinal paracellular permeability and visceral hyperalgesia, which is recapitulated in the chronic intermittent water avoidance stress (WAS) rat model. However, it is unknown whether enhanced visceral pain and permeability are intrinsically linked and correlate. Treatment of rats with lubiprostone during WAS significantly reduced WAS-induced changes in intestinal epithelial paracellular permeability and visceral hyperalgesia in a subpopulation of rats. Lubiprostone also prevented WAS-induced decreases in the epithelial tight junction protein, occludin (Ocln). To address the question of whether the magnitude of visceral pain correlates with the extent of altered intestinal permeability, we measured both end points in the same animal because of well-described individual differences in pain response. Our studies demonstrate that visceral pain and increased colon permeability positively correlate (0.6008, P = 0.0084). Finally, exposure of the distal colon in control animals to Ocln siRNA in vivo revealed that knockdown of Ocln protein inversely correlated with increased paracellular permeability and enhanced visceral pain similar to the levels observed in WAS-responsive rats. These data support that Ocln plays a potentially significant role in the development of stress-induced increased colon permeability. We believe this is the first demonstration that the level of chronic stress-associated visceral hyperalgesia directly correlates with the magnitude of altered colon epithelial paracellular permeability.

Expression of aromatase in the rostral ventromedial medulla and its role in the regulation of visceral pain.

Estrogens are known to exert a wide spectrum of actions on brain functions including modulation of pain. Besides the circulating estrogens produced mainly by the ovaries, many brain regions are also capable of de novo synthesizing estrogens, which may exert important modulatory effects on neuronal functions. This study was aimed to test the hypothesis that aromatase, the enzyme that catalyzes the conversion of testosterone to estradiols, may be distributed in the rostral ventromedial medulla (RVM), where it may impact on visceral pain. Adult female rats were treated with cyclophosphamide (CPM, 50mg/kg, ip, once every 3days) or saline. At approximately day 10 following the 3rd injection, CPM-treated rats exhibited colorectal hyperalgesia as they showed significantly greater abdominal withdrawal responses (AWR) to graded colorectal distension (CRD, 0-100mm Hg) than the saline group. Immunofluorescent staining and Western blot assay revealed that CPM-induced colorectal hyperalgesia was associated with significantly increased expression of aromatase and phosphorylated μ-type opioid receptor (pMOR) and decreased expression of total MOR in the RVM. Intracisternal application of aromatase inhibitors, fadrozole, and letrozole reversed CPM-induced colorectal hyperalgesia and restored pMOR and MOR expression in the RVM. Our observations confirmed the expression of aromatase in the RVM, a pivotal brain region in descending modulation of pain and opioid analgesia. The results support the hypothesis that locally produced estrogens in the RVM may be involved in the maintenance of chronic visceral hyperalgesia and the downstream signaling may involve phosphorylation of MOR.

Neonatal bladder inflammation induces long-term visceral pain and altered responses of spinal neurons in adult rats

Painful events early in life have been shown to increase the incidence of interstitial cystitis/painful bladder syndrome in adulthood. However, the intrinsic mechanism is not well studied. We previously reported that neonatal bladder inflammation causes chronic visceral hypersensitivity along with molecular disruption of spinal GABAergic system in rats. The present study investigates whether these molecular changes affect the integrative function and responses of bladder-sensitive primary afferent and spinal neurons. Neonatal bladder inflammation was induced by intravesicular injection of zymosan during postnatal (P) days 14–16. In adulthood (P60), the viscero-motor response (VMR) to visceral stimuli was significantly inhibited by intrathecal (i.t) HZ166 (GABAAα-2 agonist) only in neonatally saline-treated, but not in neonatally zymosan-treated rats. HZ166 significantly inhibited the responses of bladder-responsive lumbosacral (LS) spinal neurons to urinary bladder distension (UBD) and slow infusion (SI) in neonatally saline-treated rats. Similar results were also observed in naïve adult rats where HZ166 produced significant inhibition of bladder-responsive spinal neurons. However, HZ166 did not inhibit responses of UBD-responsive spinal neurons from neonatally zymosan-treated rats. The drug did not attenuate the responses of UBD-sensitive pelvic nerve afferent (PNA) fibers to UBD and SI in either group of rats tested. Immunohistochemical studies showed a significantly lower level of GABAAα-2 receptor expression in the LS spinal cord of neonatally zymosan-treated rats compared to saline-treated rats. These findings indicate that neonatal bladder inflammation leads to functional and molecular alteration of spinal GABAAα-2 receptor subtypes, which may result in chronic visceral hyperalgesia in adulthood.

Downregulation of the spinal NMDA receptor NR2B subunit during electro-acupuncture relief of chronic visceral hyperalgesia.

The involvement of spinal NR2B, a N-methyl-D-aspartate (NMDA) receptor subunit, in the therapeutic effect of electro-acupuncture (EA) on chronic visceral hyperalgesia was investigated. Chronic visceral hyperalgesia was induced using an irritable bowel syndrome (IBS) model in rats. Graded colorectal distention (CRD) stimuli at strengths of 20, 40, 60 and 80mmHg were applied, and behavioral tests were performed to measure the abdominal withdrawal reflex (AWR) in response to the CRD stimuli and assess the severity of the visceral hyperalgesia. Rats were randomly divided into four groups: normal intact (control) group, IBS model (model) group, EA-treated IBS rats (EA) group and sham EA-treated IBS rats (sham EA) group. For the EA treatment, electric stimuli were applied through needles inserted into two acupoints [Zu-san-li (ST-36) and Shang-ju-xu (ST-37)] in both hind limbs, while the sham EA treatment consisted of only the insertion of needles into these same acupoints without an application of electric stimuli. Our results showed that AWR scores of the model group responding to CRD stimuli of 20, 40, 60 and 80mmHg were significantly increased. These increased scores subsequently decreased following EA treatment (P<0.05) compared with those for the other groups. The expression of NR2B in the superficial laminae (SDH, laminae I and II), nucleus proprius (NP, laminae III and IV), neck of the dorsal horn (NECK, laminae V and VI) and central canal region (lamina X) at thoracolumbar (T13-L2) and lumbosacral (L6-S2) segmental level significantly increased in the model group versus the control group (P<0.05) and significantly decreased after EA treatment (P<0.05). There were no significant changes in neither AWR scores nor expression of the NR2B subunit in these spinal regions after the sham EA treatment. These results confirm that EA can relieve chronic visceral hyperalgesia in IBS model rats and suggest that such an effect is possibly mediated through the downregulation of the NR2B subunits of NMDA at the spinal level.

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.

Probiotic Lactobacillus rhamnosus GG (LGG) and prebiotic prevent neonatal inflammation-induced visceral hypersensitivity in adult rats.

Increasing evidence indicates a positive effect of probiotics on the nervous system. The objective of this study was to determine if probiotic Lactobacillus rhamnosus GG (LGG) and/or prebiotics polydextrose/galactooligosaccharide (PDX/GOS) can alter the colonic sensitivity in a neonatal rat model of chronic visceral hyperalgesia and to determine whether altered sensitivity is associated with changes in neurotransmitter levels in the brain. Chronic visceral hyperalgesia was induced in rats by intracolonic administration of zymosan for 3 days during postnatal day 14-16 (P14-P16). After weaning (P21), these pups were divided into groups that received either (1) control diet (CD), (2) PDX/GOS, (3) LGG, or (4) PDX/GOS + LGG. These diets were continued until visceral sensitivity was tested at P60. The viscero-motor response (VMR) to graded colorectal distension (CRD) was determined by measuring the electromyographic (EMG) activity from the abdominal external oblique muscles. The levels of neurotransmitters and biogenic amines were quantified in the frontal cortex, subcortex, brain stem, and cerebellum. At P60, the VMR to CRD in the neonatal zymosan-treated rats was significantly higher than neonatal saline-treated rats. In contrast, neonatal zymosan-treated rats that received PDX/GOS or LGG did not exhibit visceral hyperalgesia. The levels of serotonin, noradrenaline, and dopamine were significantly altered in LGG-treated rats compared to other groups. Results document that in rats LGG can attenuate neonatally induced chronic visceral pain measured in adulthood. Prolonged intake of LGG alters some key brain neurotransmitters and biogenic amines that could be involved in pain modulation.

NMDA receptor mediates chronic visceral pain induced by neonatal noxious somatic stimulation

NMDA receptors (NMDAR) are important in the development and maintenance of central sensitization. Our objective was to investigate the role of spinal neurons and NMDAR in the maintenance of chronic visceral pain. Neonatal rats were injected with acidic saline adjusted to pH 4.0 in the gastrocnemius muscle every other day for 12 days. In adult rats, NR1 and NR2B subunits were examined in the lumbo-sacral (LS) spinal cord. A baseline, visceromotor response (VMR) to graded colorectal distension (CRD) was recorded before and after administration of the NMDA antagonist, CGS-19755. Extracellular recordings were performed from CRD-sensitive LS spinal neurons and pelvic nerve afferents (PNA) before and after CGS-19755. Rats that received pH 4.0 saline injections demonstrated a significant increase in the expression NR2B subunits and VMR response to CRD>20mmHg. CGS-19755 (i.v. or i.t.) had no effect in naïve rats, but significantly decreased the response to CRD in pH 4.0 saline injected rats. CGS-19755 had no effect on the spontaneous firing of SL-A, but decreased that of SL-S. Similarly, CGS-19755 attenuates the responses of SL-S neurons to CRD, but had no effect on SL-A neurons or on the response characteristics of PNA fibers. Neonatal noxious somatic stimulation results in chronic visceral hyperalgesia and sensitizes a specific subpopulation of CRD-sensitive spinal neurons. The sensitization of these SL-S spinal neurons is attenuated by the NMDAR antagonist. The results of this study suggest that spinal NMDARs play an important role in the development of hyperalgesia early in life.

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

Predisposing effects of neonatal visceral pain on abuse-related effects of morphine in adult male Sprague Dawley rats.

Adverse early life experiences are risk factors for drug abuse and addiction. Changes in brain opioid systems have been demonstrated in response to neonatal visceral pain (NVP), but the impact of these changes on abuse-related effects of morphine are unknown. The NVP procedure used models chronic visceral hyperalgesia persisting across development. Intravenous self-administration, drug discrimination, and locomotor activity were used to compare the abuse-related effects of morphine in NVP and control rats. Rats self-administered 0.3mg/kg/inj morphine under an FR1 schedule, and dose-effect functions for morphine were then established. Separate rats were trained to discriminate 3.2mg/kg morphine from saline under an FR20 schedule, and morphine dose-effect functions were then determined in the absence and presence of 0.1mg/kg naltrexone. A third group of rats was tested with a range of morphine doses in an assay of locomotor activity, then injected daily with 10mg/kg morphine to assess locomotor sensitization. NVP rats self-administered more morphine than controls at reinforcing doses. Discriminative stimulus effects of morphine were similar between groups, but in the presence of naltrexone, the ED50 for morphine was more than 12× greater in control rats than in NVP animals. Morphine did not stimulate locomotor activity at any tested dose in NVP rats, although significant effects were observed in controls. Finally, significant locomotor sensitization was observed only in NVP rats. NVP-induced changes in brain opioid systems have persistent pharmacological consequences into adulthood and may increase sensitivity to abuse-related effects of opioids across development.