Stress-induced Visceral Hypersensitivity Research Articles

ZBTB20 mediates stress-induced visceral hypersensitivity via activating the NF-κB/transient receptor potential channel pathway.

Psychological stress is a major trigger for visceral hypersensitivity (VH) in irritable bowel syndrome. The zinc finger protein ZBTB20 (ZBTB20) is implicated in somatic nociception via modulating transient receptor potential (TRP) channels, but its role in the development of VH is unclear. This study aimed to investigate the role of ZBTB20/TRP channel axis in stress-induced VH. Rats were subjected to water avoidance stress (WAS) for 10 consecutive days. Small interfering RNA (siRNA) targeting ZBTB20 was intrathecally administered. Inhibitors of TRP channels, stress hormone receptors, and nuclear factor kappa-B (NF-κB) were administered. Visceromotor response to colorectal distension was recorded. Dorsal root ganglia (DRGs) were dissected for Western blot, coimmunoprecipitation, and chromatin immunoprecipitation. The DRG-derived neuron cell line was applied for specific research. WAS-induced VH was suppressed by the inhibitor of TRPV1, TRPA1, or TRPM8, with enhanced expression of these channels in L6-S2 DRGs. The inhibitor of glucocorticoid receptor or β2-adrenergic receptor counteracted WAS-induced VH and TRP channel expression. Concurrently, WAS-induced stress hormone-dependent ZBTB20 expression and NF-κB activation in DRGs. Intrathecally injected ZBTB20 siRNA or an NF-κB inhibitor repressed WAS-caused effect. In cultured DRG-derived neurons, stress hormones promoted nuclear translocation of ZBTB20, which preceded p65 nuclear translocation. And, ZBTB20 siRNA suppressed stress hormone-caused NF-κB activation. Finally, WAS enhanced p65 binding to the promoter of TRPV1, TRPA1, or TRPM8 in rat DRGs. ZBTB20 mediates stress-induced VH via activating NF-κB/TRP channel pathway in nociceptive sensory neurons.

Environmental enrichment reverses stress-induced changes in the brain-gut axis to ameliorate chronic visceral and somatic hypersensitivity

IntroductionBehavioral therapies, including cognitive behavioral therapy, hypnotherapy and stress management activities, have emerged as effective treatments for irritable bowel syndrome (IBS), a female predominant disorder of the brain-gut axis. IBS, affecting over 10% of the global population, typically presents with abnormal bowel habits and abdominal pain due to visceral hypersensitivity. While the mechanisms underlying how behavioral therapies treat IBS are still elusive, we had previously shown that chronic stress alters gene expression in brain regions critical for stress processing and nociception. We found that exposure to an enriched environment (EE), the rodent analogue of behavioral therapies, prior to and during the stressor was sufficient to prevent stress-induced changes in glucocorticoid receptor (GR) expression in the central nucleus of the amygdala (CeA) and hippocampus. Pre-exposure to EE also inhibited stress-induced increased colonic permeability and was able to block the induction of stress-induced visceral and somatic hypersensitivity. However, it remains unknown if EE can reverse chronic viscerosomatic hypersensitivity that persists following exposure to stress. We hypothesized that EE after chronic stress would be sufficient to reverse stress-induced changes in i) GR expression in the CeA and hippocampus, ii) ameliorate stress-induced colonic hyperpermeability and iii) restore normal visceral and somatic sensitivity in male and female rats. MethodsMale and female rats were exposed to daily water avoidance stress (WAS). After confirming the rats had developed visceral hypersensitivity, 50% of the animals were housed in EE for 2 weeks while the other 50% remained in standard housing (SH). At the end of this period, we assessed visceral and somatic sensitivity. We also collected colon tissue to measure colonic permeability. Micro-punches of tissue from the CeA and hippocampus were isolated to measure GR expression. Control animals not exposed to WAS were kept in SH for the duration of the study (n = 8 per group). ResultsIn both male and female rats, EE reversed stress-induced visceral (p < 0.001) and somatic (p < 0.01) hypersensitivity when compared to WAS animals housed in SH to levels comparable to control animals. EE exposure also reversed changes in GR expression in both the hippocampus (p < 0.01) and CeA (p < 0.01), normalizing GR expression to control levels. EE exposure ameliorated stress-induced colonic hyperpermeability in both male (p < 0.01) and female (p < 0.01) rats compared to WAS rats in SH. ConclusionOur findings suggest that behavioral therapies are viable therapeutic options for IBS as they can counter the stress-induced pathophysiology underlying IBS symptoms including visceral hypersensitivity, increased colonic permeability and altered gene expression.

Probiotic Yeast from Miso Ameliorates Stress-Induced Visceral Hypersensitivity by Modulating the Gut Microbiota in a Rat Model of Irritable Bowel Syndrome.

Recent studies indicate that probiotics, which have attracted attention as a treatment for irritable bowel syndrome, affect intestinal homeostasis. In this study, we investigated whether Zygosaccharomyces sapae (strain I-6), a probiotic yeast isolated from miso (a traditional Japanese fermented food), could improve irritable bowel syndrome symptoms. Male Wistar rats were exposed to water avoidance stress (WAS). The number of defecations during WAS and the visceral hypersensitivity before and after WAS were evaluated using colorectal distension. Tight junction changes were assessed by Western blotting. Some rats were fed with strain I-6 or β-glucan from strain I-6. Changes in the intestinal microbiota were analyzed. The effect of fecal microbiota transplantation after WAS was evaluated similarly. Caco-2 cells were stimulated with interleukin-1β and tight junction changes were investigated after coculture with strain I-6. The increased number of stool pellets and visceral hypersensitivity induced by WAS were suppressed by administering strain I-6. The decrease in tight junction protein occludin by WAS was reversed by the administration of strain I-6. β-Glucan from strain I-6 also suppressed those changes induced by WAS. In the rat intestinal microbiota, treatment with strain I-6 altered the β-diversity and induced changes in bacterial occupancy. Upon fecal microbiota transplantation, some symptoms caused by WAS were ameliorated. These results suggest that traditional fermented foods such as miso in Japan are valuable sources of probiotic yeast candidates, which may be useful for preventing and treating stress-induced visceral hypersensitivity.

Beta 3-adrenoceptor agonism ameliorates early-life stress-induced visceral hypersensitivity in male rats.

Visceral hypersensitivity, a hallmark of disorders of the gut-brain axis, is associated with exposure to early-life stress (ELS). Activation of neuronal β3-adrenoceptors (AR) has been shown to alter central and peripheral levels of tryptophan and reduce visceral hypersensitivity. In this study, we aimed to determine the potential of a β3-AR agonist in reducing ELS-induced visceral hypersensitivity and possible underlying mechanisms. Here, ELS was induced using the maternal separation (MS) model, where Sprague Dawley rat pups were separated from their mother in early life (postnatal day 2-12). Visceral hypersensitivity was confirmed in adult offspring using colorectal distension (CRD). CL-316243, a β3-AR agonist, was administered to determine anti-nociceptive effects against CRD. Distension-induced enteric neuronal activation as well as colonic secretomotor function were assessed. Tryptophan metabolism was determined both centrally and peripherally. For the first time, we showed that CL-316243 significantly ameliorated MS-induced visceral hypersensitivity. Furthermore, MS altered plasma tryptophan metabolism and colonic adrenergic tone, while CL-316243 reduced both central and peripheral levels of tryptophan and affected secretomotor activity in the presence of tetrodotoxin. This study supports the beneficial role of CL-316243 in reducing ELS-induced visceral hypersensitivity, and suggests that targeting the β3-AR can significantly influence gut-brain axis activity through modulation of enteric neuronal activation, tryptophan metabolism, and colonic secretomotor activity which may synergistically contribute to offsetting the effects of ELS.

Epigenetic mechanisms underlying stress-induced visceral pain: Resilience versus vulnerability in a two-hit model of early life stress and chronic adult stress.

Women with a history of early life stress (ELS) have a higher risk of developing irritable bowel syndrome (IBS). In addition, chronic stress in adulthood can exacerbate IBS symptoms such as abdominal pain due to visceral hypersensitivity. We previously showed that sex and the predictability of ELS determine whether rats develop visceral hypersensitivity in adulthood. In female rats, unpredictable ELS confers vulnerability and results in visceral hypersensitivity, whereas predictable ELS induces resilience and does not induce visceral hypersensitivity in adulthood. However, this resilience is lost after exposure to chronic stress in adulthood leading to an exacerbation of visceral hypersensitivity. Evidence suggests that changes in histone acetylation at the promoter regions of glucocorticoid receptor (GR) and corticotrophin-releasing factor (CRF) in the central nucleus of the amygdala (CeA) underlie stress-induced visceral hypersensitivity. Here, we aimed to investigate the role of histone acetylation in the CeA on visceral hypersensitivity in a two-hit model of ELS followed by chronic stress in adulthood. Male and female neonatal rats were exposed to unpredictable, predictable ELS, or odor only (no stress control) from postnatal days 8 to 12. In adulthood, rats underwent stereotaxic implantation of indwelling cannulas. Rats were exposed to chronic water avoidance stress (WAS, 1 h/day for 7 days) or SHAM stress and received infusions of vehicle, the histone deacetylase inhibitor trichostatin A (TSA) or the histone acetyltransferase inhibitor garcinol (GAR) after each WAS session. 24 h after the final infusion, visceral sensitivity was assessed and the CeA was removed for molecular experiments. In the two-hit model (ELS + WAS), female rats previously exposed to predictable ELS, showed a significant reduction in histone 3 lysine 9 (H3K9) acetylation at the GR promoter and a significant increase in H3K9 acetylation at the CRF promoter. These epigenetic changes were associated with changes in GR and CRF mRNA expression in the CeA and an exacerbation of stress-induced visceral hypersensitivity in female animals. TSA infusions in the CeA attenuated the exacerbated stress-induced visceral hypersensitivity, whereas GAR infusions only partially ameliorated ELS+WAS induced visceral hypersensitivity. The two-hit model of ELS followed by WAS in adulthood revealed that epigenetic dysregulation occurs after exposure to stress in two important periods of life and contributes to the development of visceral hypersensitivity. These aberrant underlying epigenetic changes may explain the exacerbation of stress-induced abdominal pain in IBS patients.

Peripheral CRF-R1/CRF-R2 antagonist, astressin C, induces a long-lasting blockade of acute stress-related visceral pain in male and female rats

Peptide CRF antagonists injected peripherally alleviate stress-induced visceral hypersensitivity (SIVH) to colorectal distension (CRD) in rodents. Here we further evaluated the dose and time-dependent inhibitory activity of several long-acting peptide CRF receptor antagonists related to astressin on SIVH, focusing on astressin C (AstC), which previously showed high efficacy on stress-related alterations of HPA axis and gut secretomotor functions. Male and female Sprague-Dawley rats pretreated subcutaneously (SC) with AstC were injected intraperitoneally (IP) with CRF 15 min later. The visceromotor responses (VMR) to graded phasic CRD (10, 20, 40 and 60 mmHg) were monitored at basal, 15 min and up to 1–8 days after pretreatment. Two other astressin analogs, hexanoyl-astressin D (Hex-AstD) and [CαMeVal19,32]-AstC, were also tested. The response to IP CRF was sex-dependent with female rats requiring a higher dose to exhibit visceral hyperalgesia. Pretreatment with AstC (30–1000 µg/kg) resulted in a dose-related inhibition of IP CRF-induced SIVH and diarrhea in both sexes. The highest dose prevented SIVH and diarrhea up to 5–7 days after a single SC injection and was lost on day 7 (females) and day 8 (males) but reinstated after a second injection of AstC on day 8 or 9 respectively. [CαMeVal19,32]-AstC and Hex-AstD (1000 µg/kg in males) also prevented SIVH. These data show the potent long-lasting anti-hyperalgesic effect of AstC in an acute model of SIVH in both male and female rats. This highlights the potential of long-acting peripheral CRF antagonists to treat stress-sensitive irritable bowel syndrome.

Epigenetic modulation of visceral nociception.

Epigenetics is a process that alters gene activity or phenotype without any changes in the underlying DNA sequence or genotype. These biological changes may have deleterious effects and can lead to various human diseases. Ongoing research is continuing to illuminate the role of epigenetics in a variety of pathophysiologic processes. Several categories of epigenetic mechanisms have been studied including chromatin remodeling, DNA methylation, histone modification, and non-coding RNA mechanisms. These epigenetic changes can have a long-term effect on gene expression without any underlying changes in the DNA sequences. The underlying pathophysiology of disorders of brain-gut interaction and stress-induced visceral pain are not fully understood and the role of epigenetic mechanisms in these disorders are starting to be better understood. Current work is underway to determine how epigenetics plays a role in the neurobiology of patients with chronic visceral pain and heightened visceral nociception. More recently, both animal models and human studies have shown how epigenetic regulation modulates stress-induced visceral pain. While much more work is needed to fully delineate the mechanistic role of epigenetics in the neurobiology of chronic visceral nociception, the current study by Louwies et al., in Neurogastroenterology and Motility provides additional evidence supporting the involvement of epigenetic alterations in the central nucleus of the amygdala in stress-induced visceral hypersensitivity in rodents.

Diosmectite treatment reduces stress-induced visceral hypersensitivity but does not affect the gut microbiota in rats.

To evaluate the effect of diosmectite on visceral hypersensitivity and intestinal transit in a rat chronic stress model and on the faecal microbiota. Wistar rats (175-225 g; n=10) were randomized into four groups: diosmectite/control, diosmectite/stress, vehicle/control, and vehicle/stress. Diosmectite (500 mg/kg, PO) was administered for five days for assessment of visceral hypersensitivity and intestinal transit and for three weeks for assessment of faecal microbiota. The stress procedure was a daily chronic passive water avoidance session. Intestinal transit was evaluated by faecal output in the hour following the last stress session. Visceral sensitivity in response to colorectal distension (CRD) was assessed at baseline and 30 min after the last stress session. In another group of rats, faecal material was collected before and after treatment with diosmectite or vehicle; genomic DNA was extracted and sequenced to characterize the faecal microbiota. Under nonstressed conditions, diosmectite treatment did not modify intestinal transit compared to the vehicle group (p=0.33). After the stress procedure, a trend towards reduction in stress-induced stool production was observed with diosmectite compared to vehicle (6.3±1.1 vs. 4.9±1.2 respectively; p=0.38). In the control condition, the number of CRD-evoked abdominal contractions was 20±4 after diosmectite and 24±2 after vehicle (p=0.75). In the stressed condition, the number of contractions increased to 34.4±2.4 after vehicle (p<0.05 compared to control). Stress-related hypersensitivity was attenuated after diosmectite treatment (26.9±2.2; p<0.05 compared to vehicle). No relevant changes were observed in the faecal microbiotic profile after treatment with diosmectite or vehicle. Diosmectite treatment attenuates stress-induced visceral hypersensitivity; this effect may contribute to the therapeutic effect of diosmectite in IBS in humans.

Early and Late Transcriptional Changes in Blood, Neural, and Colon Tissues in Rat Models of Stress-Induced and Comorbid Pain Hypersensitivity Reveal Regulatory Roles in Neurological Disease.

BackgroundIrritable bowel syndrome (IBS) and temporomandibular disorder (TMD) are two chronic pain conditions that frequently overlap in the same individual, more commonly in women. Stress is a significant risk factor, exacerbating or triggering one or both conditions. However, the mechanisms underlying IBS–TMD co-morbidity are mostly unknown.AimTo detect both specific and common stress-induced visceral hypersensitivity (SIH) and comorbid TMD–IBS pain hypersensitivity (CPH) genetic signatures over time.MethodTwenty-four female rats were randomly assigned to one of three experimental groups: naïve, SIH, and CPH (orofacial pain plus stress). RNA was extracted from blood, colon, spinal cord, and dorsal root ganglion 1 or 7 weeks after the stress paradigm. We combined differential gene expression and co-expression network analyses to define both SIH and CPH expression profiles across tissues and time.ResultsThe transcriptomic profile in blood and colon showed increased expression of genes enriched in inflammatory and neurological biological processes in CPH compared to SIH rats, both at 1 and 7 weeks after stress. In lumbosacral spinal tissue, both SIH and CPH rats compared to naïve revealed decreased expression of genes related to synaptic activity and increased expression of genes enriched in “angiogenesis,” “Neurotrophin,” and “PI3K-Akt” pathways. Compared to SIH, CPH rats showed increased expression of angiogenesis-related genes 1 week after exposure to stress, while 7 weeks post-stress the expression of these genes was higher in SIH rats. In dorsal root ganglia (DRG), CPH rats showed decreased expression of immune response genes at week 1 and inhibition of nerve myelination genes at 7 weeks compared to naïve. For all tissues, we observed higher expression of genes involved in ATP production in SIH compared to CPH at 1 week and this was reversed 7 weeks after the induction of stress.ConclusionOur study highlights an increased inflammatory response in CPH compared to SIH rats in the blood and colon. DRG and spinal transcriptomic profiles of both CPH and SIH rats showed inhibition of synaptic activity along with activation of angiogenesis. Targeting these biological processes may lead to a more profound understanding of the mechanisms underlying IBS–TMD comorbidities and new diagnostic and therapeutic strategies.

Contribution of Amygdala Histone Acetylation in Early Life Stress-Induced Visceral Hypersensitivity and Emotional Comorbidity.

Patients with irritable bowel syndrome (IBS) experience not only enhanced visceral pain but also emotional comorbidities, such as anxiety and depression. Early life stress (ELS) is a high-risk for the development of IBS. Literatures have reported an important epigenetic modulation in sustaining extrinsic phenotypes. The amygdala is closely related to the regulation of visceral functions and emotional experiences. In this study, we hypothesized that ELS-induced reprogramming inappropriate adaptation of histone acetylation modification in the amygdala may result in visceral hypersensitivity and anxiety-like behaviors in ELS rats. To test this hypothesis, the model of ELS rats was established by neonatal colorectal dilatation (CRD). Visceral hypersensitivity was assessed based on the electromyography response of the abdominal external oblique muscle to CRD. Emotional comorbidities were examined using the elevated plus maze test, open field test, and sucrose preference test. Trichostatin A (TSA) and C646 were microinjected into the central amygdala (CeA) individually to investigate the effects of different levels of histone acetylation modification on visceral hypersensitivity and emotion. We found neonatal CRD resulted in visceral hypersensitivity and anxiety-like behaviors after adulthood. Inhibiting histone deacetylases (HDACs) in the CeA by TSA enhanced visceral sensitivity but did not affect anxiety-like behaviors, whereas inhibiting HAT by C646 attenuated visceral hypersensitivity in ELS rats. Interestingly, CeA treatment with TSA induced visceral sensitivity and anxiety-like behaviors in the control rats. Western blot showed that the expressions of acetylated 9 residue of Histone 3 (H3K9) and protein kinase C zeta type (PKMζ) were higher in the ELS rats compared to those of the controls. The administration of the PKMζ inhibitor ZIP into the CeA attenuated visceral hypersensitivity of ELS rats. Furthermore, the expression of amygdala PKMζ was enhanced by TSA treatment in control rats. Finally, western blot and immunofluorescence results indicated the decrease of HDAC1 and HDAC2 expressions, but not HDAC3 expression, contributed to the enhancement of histone acetylation in ELS rats. Our results support our hypothesis that amygdala-enhanced histone acetylation induced by stress in early life results in visceral hypersensitivity and anxiety-like behaviors in ELS rats, and reversing the abnormal epigenetic mechanisms may be crucial to relieve chronic symptoms in ELS rats.

Chronic stress increases DNA methylation of the GR promoter in the central nucleus of the amygdala of female rats.

The central pathophysiological mechanisms underlying irritable bowel syndrome (IBS), a female-predominant gastrointestinal disorder characterized by abdominal pain and abnormal bowel habits, remain poorly understood. IBS patients often report that chronic stress exacerbates their symptoms. Brain imaging studies have revealed that the amygdala, a stress-responsive brain region, of IBS patients is overactive when compared to healthy controls. Previously, we demonstrated that downregulation of the glucocorticoid receptor (GR) in the central nucleus of the amygdala (CeA) underlies stress-induced visceral hypersensitivity in female rats. In the current study, we aimed to evaluate in the CeA of female rats whether chronic water avoidance stress (WAS) alters DNA methylation of the GR exon 17 promoter region, a region homologous to the human GR promoter. As histone deacetylase (HDAC) inhibitors are able to change DNA methylation, we also evaluated whether administration of the HDAC inhibitor trichostatin A (TSA) directly into the CeA prevented WAS-induced increases in DNA methylation of the GR exon 17 promoter. We found that WAS increased overall and specific CpG methylation of the GR promoter in the CeA of female rats, which persisted for up to 28days. Administration of the TSA directly into the CeA prevented these stress-induced changes of DNA methylation at the GR promoter. Our results suggest that, in females, changes in DNA methylation are involved in the regulation of GR expression in the CeA. These changes in DNA methylation may contribute to the central mechanisms responsible for stress-induced visceral hypersensitivity.

Neonatal cystitis alters mechanisms of stress-induced visceral hypersensitivity in rats

In rodent models, conditioning with acute footshock (AFS) has been demonstrated to produce bladder hypersensitivity which is more robust when rats, tested as adults, had also been pretreated with neonatal bladder inflammation (NBI). The spinal neurochemical mechanisms of pro-nociceptive processes in rats pretreated with NBI are not fully known and so the present study administered intrathecal (IT) opioid (naloxone) and NMDA receptor (MK-801) antagonists to determine whether these receptors’ actions had been altered by NBI. Female Sprague-Dawley rat pups were intravesically pretreated on postnatal days P14-P16 with a 1% zymosan solution or with control procedures and then raised to adulthood (12–15 weeks of age). Bladder hypersensitivity was induced through use of an AFS paradigm. Visceromotor responses (VMRs; abdominal muscle contractions) to graded, air pressure-controlled urinary bladder distension were used as nociceptive endpoints. Immediately following AFS pretreatments, rats were anesthetized and surgically prepared. Pharmacological antagonists were administered via an IT catheter onto the lumbosacral spinal cord and VMRs determined 15 min later. Administration of IT naloxone hydrochloride (10 μg) to rats which had been pretreated only with AFS resulted in VMRs that were more robust than VMRs in similarly pretreated rats that received IT normal saline. In contrast, IT naloxone had no significant effect on rats that had been pretreated with both NBI&AFS, although MK-801 was inhibitory. These effects of IT naloxone suggest the presence of inhibitory influences in normal rats that are absent in rats pretreated with NBI. Absence of inhibitory influences produced by AFS was also demonstrated in rats pretreated with NBI&AFS using measures of thermal paw withdrawal latency (PWL): rats pretreated with only AFS had longer PWLs than rats pretreated with both NBI&AFS. Together, a reduction in anti-nociceptive mechanisms coupled with pro-nociceptive NMDA-linked mechanisms results in more robust nociceptive responses to distension in rats which had experienced NBI.

Research Article – Neuroscience Letters Neonatal Cystitis Alters Mechanisms of Stress-Induced Visceral Hypersensitivity in Rats

Research Article – Neuroscience Letters Neonatal Cystitis Alters Mechanisms of Stress-Induced Visceral Hypersensitivity in Rats

Spinal neurochemical mechanisms of acute stress-induced visceral hypersensitivity in healthy rats

Psychological stress has been demonstrated to increase reports of pain in humans with pelvic pain of urologic origin. In rodent models, conditioning with acute footshock (AFS) has been demonstrated to increase measures of stress/anxiety as well as bladder hypersensitivity. The spinal neurochemical mechanisms of this pro-nociceptive process are unknown and so the present study administered antagonists for multiple receptors that have been associated with facilitatory mechanisms into the spinal intrathecal space. Bladder hypersensitivity was induced through use of an AFS paradigm in which female Sprague-Dawley rats received a 15-min intermittent shock treatment. Visceromotor responses (VMRs; abdominal muscle contractions) to air pressure-controlled urinary bladder distension (UBD) were used as nociceptive endpoints. Immediately following AFS treatments, rats were anesthetized (inhaled isoflurane, IP urethane) and surgically prepared. Pharmacological antagonists were administered via an intrathecal (IT) catheter onto the lumbosacral spinal cord and VMRs to graded UBD determined 15 min later. Administration of IT naloxone hydrochloride (10 μg) and IT phentolamine hydrochloride (10 μg) resulted in VMRs that were more robust than VMRs in rats that received AFS and IT normal saline whereas there was no significant effect of these drugs on VMRs in rats which underwent non-footshock procedures. In contrast, a low dose of the NMDA-receptor antagonist, MK-801 (30 μg), significantly reduced VMRs in rats made hypersensitive to UBD by AFS, but had no significant effect on rats that underwent non-footshock procedures. This study suggests that pro-nociceptive effects of AFS in otherwise healthy rats involve a spinal NMDA-linked mechanism. The effects of IT naloxone and IT phentolamine suggest the presence of inhibitory influences that are opioidergic and/or alpha-adrenergic and that are masked by the pro-nociceptive mechanisms. Other agents with no statistically significant effect on VMRs include methysergide (30 μg), ondansetron (10 μg), mecamylamine (50 μg), antalarmin (24 μg), aSVG30 (12 μg), and SSR149415 (50 μg).

A Monoclonal Anti-Calcitonin Gene-Related Peptide Antibody Decreases Stress-Induced Colonic Hypersensitivity.

Irritable bowel syndrome (IBS) is a brain-gut disorder characterized by abdominal pain and altered bowel habits. Although the etiology of IBS remains unclear, stress in adulthood or in early life has been shown to be a significant factor in the development of IBS symptomatology. Evidence suggests that aberrant calcitonin gene-related peptide (CGRP) signaling may be involved in afferent sensitization and visceral organ hypersensitivity. Here, we used a monoclonal anti-CGRP divalent antigen-binding fragment [F(ab')2] antibody to test the hypothesis that inhibition of peripheral CGRP signaling reverses colonic hypersensitivity induced by either chronic adult stress or early life stress. A cohort of adult male rats was exposed to repeated water avoidance stress. Additionally, a second cohort consisting of female rats was exposed to a female-specific neonatal odor-attachment learning paradigm of unpredictable early life stress. Colonic sensitivity was then assessed in adult animals via behavioral responses to colorectal distension (CRD). To analyze spinal nociceptive signaling in response to CRD, dorsal horn extracellular signal-regulated kinase (ERK) 1/2 phosphorylation was measured via immunohistochemistry. Repeated psychologic stress in adulthood or unpredictable stress in early life induced colonic hypersensitivity and enhanced evoked ERK1/2 phosphorylation in the spinal cord after CRD in rats. These phenotypes were reversed by administration of a monoclonal anti-CGRP F(ab')2 fragment antibody. Stress-induced changes in visceral sensitivity and spinal nociceptive signaling were reversed by inhibition of peripheral CGRP signaling, which suggests a prominent role for CGRP in central sensitization and the development of stress-induced visceral hypersensitivity. SIGNIFICANCE STATEMENT: Targeting peripheral calcitonin gene-related peptide (CGRP) with a monoclonal anti-CGRP divalent antigen-binding fragment antibody reduced central sensitization and attenuated colonic hypersensitivity induced by either chronic adult stress or early life stress. CGRP-targeting antibodies are approved for migraine prevention, and the results of this study suggest that targeting CGRP may provide a novel treatment strategy for irritable bowel syndrome-related, stress-induced visceral pain.

Human milk oligosaccharides alleviate stress-induced visceral hypersensitivity and associated microbiota dysbiosis

Pain-related functional gastrointestinal disorders (FGIDs) are characterized by visceral hypersensitivity (VHS) associated with alterations in the microbiota-gut-brain axis. Since human milk oligosaccharides (HMOs) modulate microbiota, gut and brain, we investigated whether HMOs impact VHS, and explored the role of gut microbiota. To induce VHS, C57BL/6JRj mice received hourly water avoidance stress (WAS) sessions for 10 d, or antibiotics (ATB) for 12 d. Challenged and unchallenged (Sham) animals were fed AIN93M diet (Cont) or AIN93M containing 1% of a 6-HMO mix (HMO6). VHS was assessed by monitoring the visceromotor response to colorectal distension. Fecal microbiome was analyzed by shotgun metagenomics. The effect of HMO6 sub-blends on VHS and nociceptive pathways was further tested using the WAS model. In mice fed Cont, WAS and ATB increased the visceromotor response to distension. HMO6 decreased WAS-mediated electromyographic rise at most distension volumes and overall Area Under Curve (AUC=6.12±0.50 in WAS/HMO6 vs. 9.46±0.50 in WAS/Cont; P<.0001). In contrast, VHS in ATB animals was not improved by HMO6. In WAS, HMO6 promoted most microbiota taxa and several functional pathways associated with low VHS and decreased those associated with high VHS. Among the sub-blends, 2’FL+DFL and LNT+6’SL reduced visceromotor response close to Sham/Cont values and modulated serotoninergic and CGRPα-related pathways. This research further substantiates the capacity of HMOs to modulate the microbiota-gut-brain communication and identifies mitigation of abdominal pain as a new HMO benefit. Ultimately, our findings suggest the value of specific HMO blends to alleviate pain associated FGIDs such as infantile colic or Irritable Bowel Syndrome.

Stress-induced visceral pain in female rats is associated with epigenetic remodeling in the central nucleus of the amygdala

Stress and anxiety contribute to the pathophysiology of irritable bowel syndrome (IBS), a female-predominant disorder of the gut-brain axis, characterized by abdominal pain due to heightened visceral sensitivity. In the current study, we aimed to evaluate in female rats whether epigenetic remodeling in the limbic brain, specifically in the central nucleus of the amygdala (CeA), is a contributing factor in stress-induced visceral hypersensitivity. Our results showed that 1 h exposure to water avoidance stress (WAS) for 7 consecutive days decreased histone acetylation at the GR promoter and increased histone acetylation at the CRH promoter in the CeA. Changes in histone acetylation were mediated by the histone deacetylase (HDAC) SIRT-6 and the histone acetyltransferase CBP, respectively. Administration of the HDAC inhibitor trichostatin A (TSA) into the CeA prevented stress-induced visceral hypersensitivity through blockade of SIRT-6 mediated histone acetylation at the GR promoter. In addition, HDAC inhibition within the CeA prevented stress-induced histone acetylation of the CRH promoter. Our results suggest that, in females, epigenetic modifications in the limbic brain regulating GR and CRH expression contribute to stress-induced visceral hypersensitivity and offer a potential explanation of how stress can trigger symptoms in IBS patients.

Estrogen augmented visceral pain and colonic neuron modulation in a double-hit model of prenatal and adult stress.

BACKGROUNDChronic stress during pregnancy may increase visceral hyperalgesia of offspring in a sex-dependent way. Combining adult stress in offspring will increase this sensitivity. Based on the evidence implicating estrogen in exacerbating visceral hypersensitivity in female rodents in preclinical models, we predicted that chronic prenatal stress (CPS) + chronic adult stress (CAS) will maximize visceral hyperalgesia; and that estrogen plays an important role in colonic hyperalgesia.AIMThe aim was to illuminate the role of estrogen in colonic hyperalgesia and its underlying mechanisms.METHODSWe established a CPS plus CAS rodent model in which the balloon was used to distend the colorectum. The single-fiber recording in vivo and patch clamp experiments in vitro were used to monitor the colonic neuron’s activity. The reverse transcription-polymerase chain reaction, western blot, and immunofluorescence were used to study the effects of CPS and CAS on colon primary afferent sensitivity. We used ovariectomy and letrozole to reduce estrogen levels of female rats respectively in order to assess the role of estrogen in female-specific enhanced primary afferent sensitization.RESULTSSpontaneous activity and single fiber activity were significantly greater in females than in males. The enhanced sensitization in female rats mainly came from low-threshold neurons. CPS significantly increased single-unit afferent fiber activity in L6-S2 dorsal roots in response. Activity was further enhanced by CAS. In addition, the excitability of colon-projecting dorsal root ganglion (DRG) neurons increased in CPS + CAS rats and was associated with a decrease in transient A-type K+ currents. Compared with ovariectomy, treatment with the aromatase inhibitor letrozole significantly reduced estrogen levels in female rats, confirming the gender difference. Moreover, mice treated with letrozole had decreased colonic DRG neuron excitability. The intrathecal infusion of estrogen increased brain-derived neurotrophic factor (BDNF) protein levels and contributed to the response to visceral pain. Western blotting showed that nerve growth factor protein was upregulated in CPS + CAS mice.CONCLUSIONThis study adds to the evidence that estrogen-dependent sensitization of primary afferent colon neurons is involved in the development of chronic stress-induced visceral hypersensitivity in female rats.

Amygdala microglia modify neuronal plasticity via complement C1q/C3-CR3 signaling and contribute to visceral pain in a rat model

Stress can trigger symptoms in patients with irritable bowel syndrome (IBS). Previously we demonstrated that chronic psychological stress induced microglial remodeling in the central nucleus of amygdala (CeA), and contributed to the development of visceral hypersensitivity via synaptic engulfment. However, the specific signaling mechanisms that microglia depend upon to recognize target neurons to facilitate visceral pain remain unknown. Here we test the hypothesis that the microglia in the CeA contribute to chronic stress-induced visceral hypersensitivity via complement C1q/C3-CR3 signaling-mediated synaptic remodeling. In male and female Fischer-344 rats, micropellets of corticosterone (CORT) or cholesterol (control) were stereotaxically implanted bilaterally onto the CeA. After 7 days, microglial C1q, complement receptor 3 (CR3) expression and microglia-mediated synaptic engulfment were assessed via RNAscope, quantitative PCR and immunofluorescence. The microglial inhibitor minocycline, CR3 antagonist neutrophil inhibitory factor (NIF) or vehicle were daily infused into the CeA following CORT implantations. Visceral sensitivity was assessed via a visceromotor response (VMR) to graded pressures of isobaric colorectal distension (CRD). Our results suggest that chronic exposure to elevated CORT in the CeA induced visceral hypersensitivity and amygdala microglial morphological remodeling. CORT increased microglial C1q and CR3 expression, and increased microglia-mediated synaptic engulfment. Both groups of animals with minocycline or NIF infusions reversed microglia-mediated synaptic remodeling, and attenuated CORT-induced visceral hypersensitivity. Our findings demonstrate that C1q/C3-CR3 signaling is critical for microglia-mediated synaptic remodeling in the CeA and contributes to CORT-induced visceral hypersensitivity.

Differential Activation of Colonic Afferents and Dorsal Horn Neurons Underlie Stress-Induced and Comorbid Visceral Hypersensitivity in Female Rats

Chronic Overlapping Pain Conditions, including irritable bowel syndrome (IBS) and temporomandibular disorder (TMD), represent a group of idiopathic pain conditions that likely have peripheral and central mechanisms contributing to their pathology, but are poorly understood. These conditions are exacerbated by stress and have a female predominance. The presence of one condition predicts the presence or development of additional conditions, making this a significant pain management problem. The current study was designed to determine if the duration and magnitude of peripheral sensitization and spinal central sensitization differs between restraint stress-induced visceral hypersensitivity (SIH) and chronic comorbid pain hypersensitivity (CPH; stress during pre-existing orofacial pain). SIH in female rats, as determined by the visceromotor response, persisted at least four but resolved by seven weeks. In contrast, CPH persisted at least seven weeks. Surprisingly, colonic afferents in both SIH and CPH rats were sensitized at seven weeks. CPH rats also had referred pain through seven weeks, but locally anesthetizing the colon only attenuated the referred pain through four weeks, suggesting a transition to colonic afferent independent central sensitization. Different phenotypes of dorsal horn neurons were sensitized in the CPH rats seven weeks post stress compared to four weeks or SIH rats. The current study suggests differential processing of colonic afferent input to the lumbosacral spinal cord contributes to visceral hypersensitivity during comorbid chronic pain conditions. PerspectiveChronic Overlapping Pain Conditions represent a unique challenge in pain management. The diverse nature of peripheral organs hinders a clear understanding of underlying mechanisms accounting for the comorbidity. This study highlights a mismatch between the condition-dependent behavior and peripheral and spinal mechanisms that contribute to visceral pain hypersensitivity.