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Diets consisting of refined foods (REF) are associated with poor physical (e.g., obesity and diabetes) and mental (e.g., depression) health and impaired cognition. Few animal studies have explored the causal links between diet processing and health. Instead, most studies focus on the role of macronutrients, especially carbohydrate and fat concurrently with how processed are the ingredients. We previously showed that a REF low fat diet (LFD) caused greater adiposity and impaired motivation compared to an unrefined control (CON) diet consisting of similar macronutrient ratios (Blaisdell et al., 2014). Here we test the hypothesis that the same REF LFD adversely affects attentional processes and behavioral control relative to the CON diet. Rats with ad libitum access to the REF diet for two months gained greater adiposity than rats consuming the CON diet. Rats then completed training on a vigilance task involving pressing the correct lever signaled by a brief visual cue whose onset varied across trials. A REF diet reduced accuracy when there was a delay between the start of the trial and cue onset. Poorer accuracy was due to increased premature responses, reflecting impulsivity, and omissions, indicating an inability to sustain attention. These results corroborate the links between consumption of refined foods, obesity, and poor cognition in humans. We discuss the possible causal models that underlie this link.

This study assessed functional brain activation in rats during expectation of visceral pain. Male rats were trained in step-down passive avoidance (PA) for 2 days. Upon stepping down from a platform, conditioned animals received noxious colorectal distension delivered through a colorectal balloon, whereas the balloon in control rats remained uninflated. On day 3, PA behavior was assessed while [ 14 C]-iodoantipyrine was infused intravenously, followed by immediate euthanasia. Regional cerebral blood flow-related tissue radioactivity (rCBF) was analyzed by statistical parametric mapping using 3-dimensional brains reconstructed from autoradiographic brain slice images. Associated with retrieved PA behavior, conditioned rats compared with control subjects showed increases in rCBF in sensory (anterior insula, somatosensory cortex), limbic/paralimbic regions (anterior cingulate, prelimbic cortex, amygdala), all regions previously reported to show activation during acute visceral pain. Increases in rCBF were also noted in the dorsal hippocampus, nucleus accumbens, and caudate putamen, regions associated with retrieval of PA. Organization of the underlying brain network was further delineated by functional connectivity analysis. This revealed in conditioned rats a strongly and positively connected corticostriatal cluster (cingulate, prelimbic cortex, caudate putamen). The amygdala and cerebellar hemispheres formed another positively connected cluster, which was negatively connected with the corticostriatal cluster, suggesting corticolimbic modulation. Prelimbic cortex, nucleus accumbens, and anterior insula emerged in conditioned animals as hubs. Our results show that during retrieval of PA, brain areas implicated in PA expression as well as those implicated in acute visceral pain processing were recruited, in line with findings from human brain imaging studies on pain expectation.

Irritable bowel syndrome (IBS) is characterized by chronic, recurrent abdominal pain and altered bowel habits and is currently defined by symptom criteria and the absence of detectable organic disease. The underlying pathophysiology remains incompletely understood. Despite considerable efforts by the scientific community and the pharmaceutical industry to develop novel pharmacological treatments aimed at chronic visceral pain, the traditional approach to identifying and evaluating novel drugs for this target have largely failed to translate into effective IBS treatments. However, several novel drugs aimed at normalizing bowel movements have produced clinical effects, not only on the primary target, but also on pain and discomfort. While some of the commonly used experimental animal models for the pain dimension of IBS have some face and construct validity, the predictive validity of most of the models is either unknown, or has been disappointing. A reverse translational approach is proposed, which is based on identification and characterization of brain endophenotypes in patients, followed by translation of these endophenotypes for pharmacological studies in rodent models.

Protease-activated receptors (PARs) are a family of G-protein-coupled receptors with a widespread distribution that are involved in various physiological functions including inflammation and nociception. In a recent study in Neurogastroenterology and Motility, Augé et al. describe for the first time the presence of PAR4 on visceral primary afferent neurons and its role in modulating colonic nociceptive responses, colonic hypersensitivity and primary afferent responses to PAR2 and Transient Receptor Potential Vanilloid-4 (TRPV4). Using the model of visceromotor response (VMR) to colorectal distension (CRD), they show that a PAR4 agonist delivered into the colon lumen decreases basal visceral response to CRD and reduces the exacerbated VMR to CRD induced by treatment with PAR2 or TRPV4 agonists. In isolated sensory neurons, they show that a PAR4 agonist inhibits calcium mobilization induced by PAR2 or TRPV4 agonists. Finally, they describe increased pain behaviour evoked by luminal application of mustard oil in PAR4 deficient mice compared to wild type controls. The newly discovered role of PAR4 in modulating visceral pain adds to our growing understanding of the contribution of colonic proteases and PARs to the mechanisms involved in colonic hypersensitivity and their potential role as therapeutic targets for irritable bowel syndrome.

Studies in healthy human subjects and patients with irritable bowel syndrome suggest sex differences in cerebral nociceptive processing. Here we examine sex differences in functional brain activation in the rat during colorectal distention (CRD), a preclinical model of acute visceral pain. [(14)C]-iodoantipyrine was injected intravenously in awake, non-restrained female rats during 60- or 0-mmHg CRD while electromyographic abdominal activity (EMG) and pain behavior were recorded. Regional cerebral blood flow-related tissue radioactivity was analyzed by statistical parametric mapping from autoradiographic images of three-dimensionally reconstructed brains. Sex differences were addressed by comparing the current data with our previously published data collected from male rats. While sex differences in EMG and pain scores were modest, significant differences were noted in functional brain activation. Females showed widespread changes in limbic (amygdala, hypothalamus) and paralimbic structures (ventral striatum, nucleus accumbens, raphe), while males demonstrated broad cortical changes. Sex differences were apparent in the homeostatic afferent network (parabrachial nucleus, thalamus, insular and dorsal anterior cingulate cortices), in an emotional-arousal network (amygdala, locus coeruleus complex), and in cortical areas modulating these networks (prefrontal cortex). Greater activation of the ventromedial prefrontal cortex and broader limbic/paralimbic changes in females suggest greater engagement of affective mechanisms during visceral pain. Greater cortical activation in males is consistent with the concept of greater cortical inhibitory effects on limbic structures in males, which may relate to differences in attentional and cognitive attribution to visceral stimuli. These findings show remarkable similarities to reported sex differences in brain responses to visceral stimuli in humans.

Preclinical drug development for visceral pain has largely relied on quantifying pseudoaffective responses to colorectal distension (CRD) in restrained rodents. However, the predictive value of changes in simple reflex responses in rodents for the complex human pain experience is not known. Male rats were implanted with venous cannulas and with telemetry transmitters for abdominal electromyographic (EMG) recordings. [(14)C]-iodoantipyrine was injected during noxious CRD (60 mmHg) in the awake, nonrestrained animal. Regional cerebral blood flow (rCBF)-related tissue radioactivity was quantified by autoradiography and analyzed in the three-dimensionally reconstructed brain by statistical parametric mapping. 60-mmHg CRD, compared with controls (0 mmHg) evoked significant increases in EMG activity (267+/-24% vs. 103+/-8%), as well as in behavioral pain score (77+/-6% vs. 3+/-3%). CRD elicited significant increases in rCBF as expected in sensory (insula, somatosensory cortex), and limbic and paralimbic regions (including anterior cingulate cortex and amygdala). Significant decreases in rCBF were seen in the thalamus, parabrachial nucleus, periaqueductal gray, hypothalamus and pons. Correlations of rCBF with EMG and with behavioral pain score were noted in the cingulate, insula, lateral amygdala, dorsal striatum, somatosensory and motor regions. Our findings support the validity of measurements of cerebral perfusion during CRD in the freely moving rat as a model of functional brain changes in human visceral pain. However, not all regions demonstrating significant group differences correlated with EMG or behavioral measures. This suggests that functional brain imaging captures more extensive responses of the central nervous system to noxious visceral distension than those identified by traditional measures.

Visceral hypersensitivity has been implicated as an important pathophysiological mechanism in functional gastrointestinal disorders. In this study, we investigated whether the sustained visceral hyperalgesia induced by repeated psychological stress in rats involves the activation of CRF1 signaling system using two different antagonists. Male Wistar rats were exposed to 10 consecutive days of water avoidance stress (WAS) or sham stress for 1 h/day, and the visceromotor response to phasic colorectal distension (CRD) was assessed before and after the stress period. Animals were injected subcutaneously with the brain penetrant CRF1 antagonist, CP-154,526, acutely (30 min before the final CRD) or chronically (via osmotic minipump implanted subcutaneously, during stress) or with the peripherally restricted, nonselective CRF1 and CRF2 antagonist, astressin, chronically (15 min before each stress session). Repeated WAS induced visceral hypersensitivity to CRD at 40 and 60 mmHg. CP-154,526 injected acutely significantly reduced stress-induced visceral hyperalgesia at 40 mmHg but not at 60 mmHg. Chronic subcutaneous delivery of astressin reduced the stress-induced visceral hyperalgesia to baseline at all distension pressures. Interestingly, chronically administered CP-154,526 eliminated hyperalgesia and produced responses below baseline at 40 mmHg and 60 mmHg, indicating a hypoalgesic effect of the compound. These data support a major role for CRF1 in both the development and maintenance of visceral hyperalgesia induced by repeated stress and indicate a possible role of peripheral CRF receptors in such mechanisms.

Despite its beneficial effect in IBS patients, the mechanism of action of the 5-HT3 receptor (5-HT3R) antagonist alosetron is still incompletely understood. We aimed to characterize the effect and site(s) of action in a model of stress-induced sensitization of visceral nociception in rats. Adult male Wistar rats were equipped for recording of visceromotor response (VMR) to phasic colorectal distension (CRD; 10-60 mmHg). VMR to CRD was recorded 24 h after an acute session of water avoidance (WA) stress (post-WA). Baseline and post-WA responses were measured in rats exposed to WA or sham-WA, treated with alosetron at 0.3 mg/kg subcutaneously (s.c.) 25 nmol intrathecally (i.t.) or vehicle before post-WA CRD. Some rats were treated with capsaicin/vehicle on the cervical vagus nerve and received alosetron (0.3 mg/kg, s.c.) 15 min before post-WA CRD. WA stress led to visceral hyperalgesia 24 h later. Alosetron (0.3 mg/kg, s.c.), failed to inhibit WA-induced exacerbation of VMR to CRD. Stress-induced visceral hyperalgesia was abolished when alosetron was injected intrathecally (P

The gastrointestinal tract in humans is host to 1 × 1013 to 1 × 1014 microorganisms amongst which commensal bacteria and pathogenic bacteria form a symbiotic environment (Frosali et al. 2015). This homeostatic state is key to the maintenance of the health of the host, and the scientific community is increasingly interested in investigating the effect of disruption of this balance, also called dysbiosis, in disease. In the recent years important findings have highlighted the influence of gut microbiota on the development and function of the nervous system and the state of the immune system, as seen in inflammatory bowel diseases (Maranduba et al. 2015). One of the greatest challenges in investigating the microbiome composition and function in human health and disease is to determine the causality between microbiome variation and pathophysiology. The gut microbiota and bacterial metabolites have recently been the focus of intense research looking at the molecular mechanisms underlying their role in the regulation of the neuroimmune system and physiological functions, and progress in the field has been facilitated by rapidly evolving bioanalytical techniques. Multiple clinical studies have focused on evaluating the composition and organization of gut microbiota and related metabolites in patients suffering from irritable bowel syndrome (IBS) compared with healthy controls. This research effort is driven by the need to shed light on the relationship between diet and the composition of the microbiome, and IBS symptoms, which include abdominal pain. Overall, the findings have been consistent in showing a shift in fecal microbial community composition in IBS (Rajilic-Stojanovic et al. 2015). What is less straightforward is the specificity, nature and direction of those microbial differences across the different studies, which seem to be affected by variables such as the analytical methods used, geography, diet, or homogeneity of the IBS groups (Mayer et al. 2014). However, based on several pre-clinical and clinical studies, it seems that treatments targeting the gut microbiome, using probiotics or antibiotics in adults, may offer benefits in terms of symptoms and, in particular, symptoms of visceral pain (Mayer et al. 2014). Studies in pediatric IBS populations have reported a beneficial effect of Lactobacillus rhamnosus on pain (Horvath et al. 2011), and Lactobacillus reuteri DSM 17938 (DSM) was found to be effective at reducing symptoms of infantile colic in breast-fed infants (Savino et al. 2010, Guandalini, 2014). Despite the increasing number of reports showing a modulatory effect of manipulating the gut microbiome on gastrointestinal function including pain, motility or permeability, the mechanisms by which the gut microbiota takes part in brain–gut activity, as in a microbiome–brain–gut system, remain unclear. With multiple mechanisms likely to be at play, preclinical studies have been instrumental in identifying the layers of those signalling mechanisms. In this issue of The Journal of Physiology, Perez-Burgos et al. (2015) examine the potential role of the transient receptor potential vanilloid 1 (TRPV1) channel in the mechanisms mediating an antinociceptive effect of L. reuteri DSM17938 (DSM) in a rat model of visceral pain induced by gastric distension. TRPV1 receptors are key in the transduction of pain signals in the gut (Blackshaw 2014) and constitute a rational target to look at when pursuing potential mechanisms of action of agents with a modulatory effect on visceral pain. This is further supported by previous evidence showing increased TRPV1 expression in rectal biopsies from IBS patients (Akbar et al. 2008). By testing the effect of DSM on capsaicin-evoked rise in intracellular Ca2+ in isolated DRG neurons from mice, Perez-Burgos et al. were able to show an inhibitory effect of DSM on TRPV1 channels in extrinsic primary sensory terminals. The fact that this effect was reproduced by conditioned medium but not gamma-irradiated dead DSM suggests that the effect of DSM may be related to a product derived from the bacterium, though this was not investigated in this study. This is an important consideration for the future as the identification of bacterium-derived products may help in characterizing the candidate signalling molecules mediating the effect of DSM on TRPV1 or, more generally, mediating the effect of gut bacteria on brain–gut communication. Another important observation from the current study is that a different probiotic bacterium, JB-1, previously shown to reduce pain-related pseudoaffective responses and spinal nerve single fibre firing induced by gut distension, failed to reduce the capsaicin evoked Ca2+ rise in DRG neurons seen with DSM treatment. These results indicate that different bacteria used in the context of preclinical models of gut distension most likely act via different signalling mechanisms to modulate pain response. One should also consider the role played by methodological differences in the contrasting results observed across the literature and reflect on the need to define new standards for the design of translational preclinical studies. This should also be applied to clinical studies for which methodological differences may account for the heterogeneity of findings on the efficacy of manipulating the gut microbiome on gastrointestinal symptoms. Despite these limitations, there is no doubt that mechanistic studies like the one presented in the paper from Perez-Burgos et al. have a great implication for the future development of new therapeutic tools targeting the gut microbiome for the treatment of gastrointestinal diseases.

Psychological stress exacerbates interstitial cystitis/bladder pain syndrome (IC/BPS), a lower urinary tract pain disorder characterized by increased urinary frequency and bladder pain. Glutamate (Glu) is the primary excitatory neurotransmitter modulating nociceptive networks. Glt1, an astrocytic transporter responsible for Glu clearance, is critical in pain signaling termination. We sought to examine the role of Glt1 in stress-induced bladder hyperalgesia and urinary frequency. In a model of stress-induced bladder hyperalgesia with high construct validity to human IC/BPS, female Wistar-Kyoto (WKY) rats were subjected to 10-day water avoidance stress (WAS). Referred hyperalgesia and tactile allodynia were assessed after WAS with von Frey filaments. After behavioral testing, we assessed Glt1 expression in the spinal cord by immunoblotting. We also examined the influence of dihydrokainate (DHK) and ceftriaxone (CTX), which downregulate and upregulate Glt1, respectively, on pain development. Rats exposed to WAS demonstrated increased voiding frequency, increased colonic motility, anxiety-like behaviors, and enhanced visceral hyperalgesia and tactile allodynia. This behavioral phenotype correlated with decreases in spinal Glt1 expression. Exogenous Glt1 downregulation by DHK resulted in hyperalgesia similar to that following WAS. Exogenous Glt1 upregulation via intraperitoneal CTX injection inhibited the development of and reversed preexisting pain and voiding dysfunction induced by WAS. Repeated psychological stress results in voiding dysfunction and hyperalgesia that correlate with altered central nervous system glutamate processing. Manipulation of Glu handling altered the allodynia developing after psychological stress, implicating Glu neurotransmission in the pathophysiology of bladder hyperalgesia in the WAS model of IC/BPS.

Author(s): Bradesi, Sylvie; Karagiannides, Iordanes; Bakirtzi, Kyriaki; Joshi, Swapna Mahurkar; Koukos, Georgios; Iliopoulos, Dimitrios; Pothoulakis, Charalabos; Mayer, Emeran A | Abstract: 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.

In rodents, maternal pup interactions play an important role in programming the stress responsiveness of the adult organism. The aims of this study were 1) to determine the effect of different neonatal rearing conditions on acute and delayed stress-induced visceral sensitivity as well as on other measures of stress sensitivity of the adult animal; and 2) to determine the role of corticotropin-releasing factor receptor (CRF-R) subtype 1 (CRF1R) in mediating visceral hypersensitivity. Three groups of male Long-Evans rat pups were used: separation from their dam for 180 min daily from postnatal days 2–14 (MS180), daily separation (handling) for 15 min (H), or no handling. The visceromotor responses (VMR) to colorectal distension, stress-induced colonic motility, and anxiety-like behavior were assessed in the adult rats. The VMR was assessed at baseline, immediately after a 1-h water avoidance (WA) stress, and 24 h poststress. Astressin B, a nonselective CRF-R antagonist, or CP-154,526, a selective CRF1R antagonist, was administered before the stressor and/or before the 24-h measurement. MS rats developed acute and delayed stress-induced visceral hyperalgesia. In contrast, H rats showed hypoalgesia immediately after WA and no change in VMR on day 2. MS rats with visceral hyperalgesia also exhibited enhanced stress-induced colonic motility and increased anxiety-like behavior. In MS rats, both CRF-R antagonists abolished acute and delayed increases in VMR. Rearing conditions have a significant effect on adult stress responsiveness including immediate and delayed visceral pain responses to an acute stressor. Both acute and delayed stress-induced visceral hypersensitivity in MS rats are mediated by the CRF/CRF1R system.

Objective To evaluate whether anxiety-prone rats exposed to chronic water avoidance stress (WAS) develop visceral bladder hyperalgesia in addition to increased voiding frequency and anxiety-related behaviors. Materials and Methods Female Wistar-Kyoto (WKY) rats were exposed to chronic (10-day) WAS or sham paradigms. Referred hyperalgesia and tactile allodynia were tested using von Frey filaments applied to the suprapubic region and plantar region of the hindpaw, respectively. To confirm that suprapubic nociception represented referred visceral bladder hyperalgesia, we recorded abdominal visceromotor responses (VMR) to slow (100 μl/min) and fast (1 cc/sec) bladder filling with room temperature or ice-cold saline. We assessed the development of hyperalgesia over the 10-day WAS protocol and the durability of increased pain sensations over time. Results Animals exposed to chronic WAS had significantly lower hindpaw withdrawal thresholds post-stress and significant differences in referred hyperalgesia. Rats exposed to chronic WAS demonstrated an increased pain response to suprapubic stimulation and decreased response threshold to mechanical hindpaw stimulation by day 8 of the stress protocol, which persisted for more than one month. Animals exposed to chronic WAS showed increased VMR to fast filling and ice water testing in comparison to sham animals. Cystometry under anesthesia did not show increases in the frequency of non-voiding contractions. Conclusion Chronic WAS induces sustained bladder hyperalgesia, lasting over a month after exposure to stress. The urinary frequency demonstrated previously in anxiety-prone rats exposed to chronic WAS seems to be associated with bladder hyperalgesia, suggesting that this is a potential model for future studies of bladder hypersensitivity syndromes such as interstitial cystitis/painful bladder syndrome (IC/PBS).

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 ([(14)C]-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.

Background/Aims: Spinal glia activation has been proposed as one mechanism underlying visceral hyperalgesia in a rodent model of chronic stress. In order to assess the possible role of changes in circulating cytokines and in blood-spinal cord barrier (BSCB) permeability in spinal glia activation, we studied the time course of peripheral and spinal pro-inflammatory cytokines and of spinal and satellite glia markers in response to repeated water avoidance (WA) stress. Methods: Spinal cords and dorsal root ganglion cells (DRGs) were collected from control rats, rats exposed to 1-hour WA, or 1-hour WA daily for 5 days or 1-hour WA daily for 10 days. Results: We demonstrated a time-dependent change in circulating IL-1β and spinal IL-1β, IL-6 and TNF-α in stressed animals compared with controls. We found altered expression of the astrocyte markers GFAP and Connexin 43 in spinal and DRG samples at different time points. Finally, WA was associated with increased BSCB permeability. Conclusions: These findings confirm the concept that both peripheral and spinal immune markers are altered after chronic WA and suggest a possible link between stress-induced increase of peripheral pro-inflammatory cytokines, changes in satellite glial cells, increase in BSCB permeability and increase in spinal pro-inflammatory mediators suggesting glia activation.

The majority of functional gastrointestinal disorders are characterised by recurrent abdominal pain, with stress playing an important role in first onset and exacerbation of existing symptoms. These disorders are currently defined by symptom criteria, while their pathophysiology remains controversial and incompletely understood. Modeling these disorders in humans and animals has been difficult. While some of the models have adequate face and construct validity, the predictive validity of most of the models has been disappointing, which has put into question the traditional modeling approach. Similar problems have been encountered in drug development for pain and psychiatric disorders. New approaches have been proposed in the form of reverse translation, which include better characterisation of biological intermediate phenotypes in human disease which can be modeled in humans and in animals. Continuation of the current approach focusing on complex clinical phenotypes is likely to be ineffective for the development of novel and effect treatments.

BACKGROUND Linaclotide is a novel, orally administered investigational drug currently in clinical development for the treatment of constipation-predominant irritable bowel syndrome (IBS-C) and chronic idiopathic constipation. Visceral hyperalgesia is a major pathophysiological mechanism in IBS-C. Therefore, we investigated the anti-nociceptive properties of linaclotide in rodent models of inflammatory and non-inflammatory visceral pain and determined whether these pharmacological effects are linked to the activation of guanylate cyclase C (GC-C). METHODS Orally administered linaclotide was evaluated in non-inflammatory acute partial restraint stress (PRS) and acute water avoidance stress (WAS) models in Wistar rats, and in a trinitrobenzene sulfonic acid (TNBS)-induced inflammatory model in Wistar rats and GC-C null mice. KEY RESULTS In TNBS-induced colonic allodynia, linaclotide significantly decreased the number of abdominal contractions in response to colorectal distension without affecting the colonic wall elasticity change in response to distending pressures after TNBS. However, linaclotide had no effect on visceral sensitivity under basal conditions. In addition, linaclotide significantly decreased colonic hypersensitivity in the PRS and WAS models. In wild type (wt) and GC-C null mice, the instillation of TNBS induced similar hyperalgesia and allodynia. However, in post-inflammatory conditions linaclotide significantly reduced hypersensitivity only in wt mice, but not in GC-C null mice. CONCLUSIONS & INFERENCES These findings indicate that linaclotide has potent anti-nociceptive effects in several mechanistically different rodent models of visceral hypersensitivity and that these pharmacological properties of linaclotide are exerted through the activation of the GC-C receptor. Therefore, linaclotide may be capable of decreasing abdominal pain in patients suffering from IBS-C.

Visceral hypersensitivity and stress have been implicated in the pathophysiology of functional gastrointestinal disorders. We used a selective vasopressin 3 (V3) receptor antagonist SSR149415 to investigate the involvement of the vasopressin (AVP)/V3 signaling system in the development of stress-induced visceral hyperalgesia in rats. Rats were exposed to a daily 1-h session of water avoidance stress (WAS) or sham WAS for 10 consecutive days. The visceromotor response to phasic colorectal distension (CRD, 10–60 mmHg) was assessed before and after stress. Animals were treated daily with SSR149415 (0.3, 1, or 3 mg/kg ip 30 min before each WAS or sham WAS session), with a single dose of SSR149415 (1 mg/kg ip), or the selective corticotropin-releasing factor 1 (CRF1) antagonist DMP-696 (30 mg/kg po) before CRD at day 11. Effects of a single dose of SSR149415 (10 mg/kg iv) on acute mechanical sensitization during repetitive CRD (12 distensions at 80 mmHg) were also assessed. In vehicle-treated rats, repeated WAS increased the response to CRD, indicating visceral hypersensitivity. Repeated administration of SSR149415 at 1 or 3 mg/kg completely prevented stress-induced visceral hyperalgesia. Similarly, a single dose of DMP-696 or SSR149415 completely blocked hyperalgesic responses during CRD. In contrast, a single dose of SSR149415 did not affect the acute hyperalgesic responses induced by repeated, noxious distension. These data support a major role for V3 receptors in repeated psychological stress-induced visceral hyperalgesia and suggest that pharmacological manipulation of the AVP/V3 pathway might represent an attractive alternative to the CRF/CRF1 pathway for the treatment of chronic stress-related gastrointestinal disorders.

Irritable bowel syndrome (IBS) remains an incompletely understood, common syndrome with significant unmet medical needs. Significant progress has been made in the development of novel therapies aimed at normalizing bowel habit alterations and abdominal discomfort, even though some of the most effective treatments are currently only available for patients under a restricted access program from the FDA. Preclinical evidence supports the potential usefulness of several compounds in development for the treatment of chronic abdominal pain. Recent new evidence for a possible role of altered microflora and altered host microbial interactions may provide new treatment targets in the future.

Despite considerable efforts by academic researchers and by the pharmaceutical industry, the development of novel pharmacological treatments for irritable bowel syndrome (IBS) and other functional gastrointestinal (GI) disorders has been slow and disappointing. The traditional approach to identifying and evaluating novel drugs for these symptom-based syndromes has relied on a fairly standard algorithm using animal models, experimental medicine models and clinical trials. In the current article, the empirical basis for this process is reviewed, focusing on the utility of the assessment of visceral hypersensitivity and GI transit, in both animals and humans, as well as the predictive validity of preclinical and clinical models of IBS for identifying successful treatments for IBS symptoms and IBS-related quality of life impairment. A review of published evidence suggests that abdominal pain, defecation-related symptoms (urgency, straining) and psychological factors all contribute to overall symptom severity and to health-related quality of life. Correlations between readouts obtained in preclinical and clinical models and respective symptoms are small, and the ability to predict drug effectiveness for specific as well as for global IBS symptoms is limited. One possible drug development algorithm is proposed which focuses on pharmacological imaging approaches in both preclinical and clinical models, with decreased emphasis on evaluating compounds in symptom-related animal models, and more rapid screening of promising candidate compounds in man.

Background & Aims: The neurokinin 1 receptors (NK 1 Rs) and substance P (SP) have been implicated in the stress and/or pain pathways involved in chronic pain conditions. Here we examined the participation of NK 1 Rs in sustained visceral hyperalgesia observed in rats exposed to chronic psychological stress. Methods: Male Wistar rats were exposed to daily 1-hour water avoidance stress (WA) or sham WA for 10 consecutive days. We tested intraperitoneal or intrathecal injection of the NK 1 R antagonist SR140333 on the visceromotor reflex to colorectal distention in both groups at day 11. Real-time reverse-transcription polymerase chain reaction, Western blot, and immunohistochemistry were used to assess the expression of NK 1 Rs and/or SP in samples of colon, spinal cord, and dorsal root ganglia. Results: Both intraperitoneal and intrathecal SR140333 injection diminished the enhanced visceromotor reflex to colorectal distention at day 11 in stressed rats but did not affect the response in control animals. Real-time polymerase chain reaction and Western blotting demonstrated stress-induced up-regulation of spinal NK 1 Rs. Immunohistochemistry showed an increased number of NK 1 R-expressing neurons in the laminae I of the dorsal horn in stressed rats. The expression of NK 1 Rs was decreased in colon from stressed rats compared with control. The expression of SP gene precursor in dorsal root ganglia was unchanged in stressed rats compared with controls. Conclusions: Stress-induced increased NK 1 R expression on spinal neurons and the inhibitory effect of intrathecal NK 1 R antagonist on visceral hyperalgesia support the key contribution of spinal NK 1 Rs in the molecular pathways involved in the maintenance of visceral hyperalgesia observed after chronic WA.

Chronic stress plays an important role in the development and exacerbation of symptoms in functional gastrointestinal disorders. To better understand the mechanisms underlying this relationship, we aimed to characterize changes in visceral and somatic nociception, colonic motility, anxiety-related behavior, and mucosal immune activation in rats exposed to 10 days of chronic psychological stress. Male Wistar rats were submitted daily to either 1-h water avoidance (WA) stress or sham WA for 10 consecutive days. The visceromotor response to colorectal distension, thermal somatic nociception, and behavioral responses to an open field test were measured at baseline and after chronic WA. Fecal pellets were counted after each WA stress or sham WA session as a measure of stress-induced colonic motility. Colonic samples were collected from both groups and evaluated for structural changes and neutrophil infiltration, mast cell number by immunohistochemistry, and cytokine expression by quantitative RT-PCR. Rats exposed to chronic WA (but not sham stress) developed persistent visceral hyperalgesia, whereas only transient changes in somatic nociception were observed. Chronically stressed rats also exhibited anxiety-like behaviors, enhanced fecal pellet excretion, and small but significant increases in the mast cell numbers and the expression of IL-1β and IFN-γ. Visceral hyperalgesia following chronic stress persisted for at least a month. Chronic psychological stress in rats results in a robust and long-lasting alteration of visceral, but not somatic nociception. Visceral hyperalgesia is associated with other behavioral manifestations of stress sensitization but was only associated with minor colonic immune activation arguing against a primary role of mucosal immune activation in the maintenance of this phenomenon.

Hypersensitivity to rectal or colonic distension characterizes most patients with IBS and increased gut permeability has been described in post-dysenteric IBS patients. However, no link has been established between these two events. The aim of this study was to determine (i) whether chemical blockade of stress-induced increase of colonic paracellular permeability by 2,4,6 triaminopyrimidine (TAP) affects the concomitant hypersensitivity to colonic distension, (ii) the role of epithelial cell contraction in the stress-induced increased permeability and hyperalgesia, using a myosin light chain kinase inhibitor (ML-7). The effect of acute partial restraint stress (PRS) on visceral sensitivity to colorectal distension (RD) was assessed by abdominal muscle electromyography. Colonic paracellular permeability was determined by measuring percentage of urinary 51Cr-EDTA recovery after intracolonic infusion. The effect of stress on both parameters was evaluated after TAP, ML-7 or vehicle pretreated animals. PRS significantly increased colonic paracellular permeability and the number of spike bursts for all volumes of RD applied compared to sham. TAP suppressed the stress-induced increase of colonic paracellular permeability and sensitivity to colonic distension. Similarly, ML-7 blocked the stress-induced increase of colonic paracellular permeability and sensitivity. Neither ML-7 nor TAP had any effect on both permeability and sensitivity in absence of stress. The increase of colonic permeability induced by PRS results from epithelial cell cytoskeleton contraction through myosin light chain kinase activation and this increase is responsible for stress-induced rectal hypersensitivity.

The mechanism(s) underlying stress-induced colonic hypersensitivity (SICH) are incompletely understood. Our aims were to assess the acute and delayed (24 h) effect of water avoidance (WA) stress on visceral nociception in awake male Wistar rats and to evaluate the role of two stress-related modulation systems: the substance P/neurokinin-1 receptor (SP/NK1R) and the corticotropin-releasing factor (CRF)/CRF1 receptor (CRF/CRF1R) systems, as well as the possible involvement of the sympathetic nervous system. Visceral pain responses were measured as the visceromotor response to colorectal distension (CRD) at baseline, immediately after WA and again 24 h later. The NK1R antagonists RP-67580 and SR-140333 and the CRF1R antagonist CP-154526 were injected 15 min before WA or 1 h before the CRD on day 2. Chemical sympathectomy was performed by repeated injection of 6-hydroxydopamine. WA stress resulted in a significant increase in the visceromotor response on day 2, but no change immediately after WA. Injection of CP-154526 abolished delayed SICH when applied either before WA stress or before the CRD on day 2. Both NK1R antagonists only decreased SICH when injected before the CRD on day 2. Chemical sympathectomy did not affect delayed SICH. Our results indicate that in male Wistar rats, both NK1R and CRF1R activation, but not sympathetic nervous system activation, play a role in the development of SICH.

Hormonal cycling may be related to a higher incidence of pain syndrome in female. As tachykinins are pivotal in stress-induced colonic dysfunction, we investigated whether ovarian steroids influence stress-induced visceral hypersensitivity to rectal distension (RD) in female rats and further, whether this influence involves NK1 receptors. Female Wistar rats, either intact or ovariectomized (OVX), were equipped for abdominal muscle electromyography and submitted to 2-h partial restraint stress (PRS) or sham-PRS. First, the effect of PRS was evaluated in intact rats. Second, abdominal response to RD was recorded in OVX rats treated with either, progesterone, 17beta-estradiol, 17beta-estradiol-plus-progesterone, or vehicle, in both basal and PRS conditions. Third, the NK1 receptor-antagonist, SR140333, was tested in PRS-intact and PRS-OVX rats under 17beta-estradiol or 17beta-estradiol-plus-progesterone treatment. PRS induced visceral hypersensitivity to RD and this effect was prevented by ovariectomy. OVX rats treated with 17beta-estradiol or 17beta-estradiol-plus-progesterone, but not progesterone alone, exhibited visceral hypersensitivity after PRS similar to that of intact rats. Both stress-induced visceral hypersensitivity in intact rats and the hormonally-restored visceral hyper-responsiveness of OVX rats were antagonized by SR140333. It is concluded, therefore, that stress-induced visceral hypersensitivity in female rats is estrogens-dependent and mediated through NK1 receptor activation.

Objective Psychological stress plays a role in the exacerbation of functional lower urinary tract disorders, such as painful bladder syndrome and overactive bladder. To better understand the mechanism underlying this relationship, we characterized changes in micturition, anxiety-related behavior, and bladder pathology in rats exposed to repeated water avoidance (WA) stress. Methods Twenty-four Wistar rats were subjected to WA stress or sham. Immediately after acute (day 1) and chronic (day 10) stress or sham, rats were placed in a metabolic cage for a 2-hour voiding behavior assessment. Voiding parameters were compared with baseline values obtained before stress. Four animals from each group were sacrificed on day 10 and bladders harvested for histologic and gene expression studies. The remaining 8 animals per group underwent repeated voiding assessment every 3 days for 1 month followed by 10 days of repeat WA stress or sham. Bladder histology and gene expression were studied. Results Rats exposed to WA stress developed a significant increase in micturition frequency and decrease in latency to void, voiding interval, and volume of first void compared with sham and baseline. Alterations in micturition persisted for approximately 1 month. Stressed rats showed increased fecal pellet excretion and anxiety-like behavior. In addition, bladder specimens from stressed animals revealed increased angiogenesis, and increased total and activated mast cells. Conclusion In rats, repeated psychological stress results in lasting alterations in micturition frequency, interval, and volume. This rodent model may represent a valid tool for studying syndromes characterized by increased urinary frequency.

Background & Aims Chronic psychological stress is associated with visceral hyperalgesia and increased expression of spinal NK1 receptors (NK1Rs). We aimed to identify the role of spinal microglia in this process. Methods Male Wistar rats were exposed to water avoidance (WA) or sham stress 1 hour each day for 10 days and given daily injections of minocycline, the p38 inhibitor SB203580, or saline. Phosphorylation levels of the kinase p38 (P-p38), the microglia marker OX42, NK1R, and IκBα were assessed by immunoblotting and/or immunostaining of spinal samples collected at day 11. The visceromotor response to colorectal distention at baseline and following WA were also assayed in rats given injections of minocycline, SB203580, or vehicle. The effects of fractalkine were assessed on the visceromotor response in rats exposed to minocycline or vehicle. Results P-p38 protein levels and immunoreactivity were increased in stressed rats and colocalized with OX42-positive cells and neurons in the dorsal horn. This increase was reversed by minocycline or SB203580 exposure. Stress-induced increased NK1R expression was blocked by minocycline but not SB203580. WA-induced decreased IκBα expression was blocked by minocycline and SB203580. WA-induced hyperalgesia was blocked by minocycline and SB203580 intrathecally. Fractalkine-induced hyperalgesia was blocked by minocycline. Conclusions This is the first demonstration that stress-induced activation of spinal microglia has a key role in visceral hyperalgesia and associated spinal NK1R up-regulation.