Intravenous Polyethylene Glycol Alleviates Intestinal Ischemia-Reperfusion Injury in a Rodent Model.

Intestinal ischemia-reperfusion injury (IRI) is a common clinical entity, and its outcome is unpredictable due to the triad of inflammation, increased permeability and bacterial translocation. Polyethylene glycol (PEG) is a polyether compound that is extensively used in pharmacology as an excipient in various products. More recently, this class of products have shown to have potent anti-inflammatory, anti-apoptotic, immunosuppressive and cell-membrane-stabilizing properties. However, its effects on the outcome after intestinal IRI have not yet been investigated. We hypothesized that PEG administration would reduce the effects of intestinal IRI in rodents. In a previously described rat model of severe IRI (45 min of ischemia followed by 60 min of reperfusion), we evaluated the effect of IV PEG administration at different doses (50 and 100 mg/kg) before and after the onset of ischemia. In comparison to control animals, PEG administration stabilized the endothelial glycocalyx, leading to reduced reperfusion edema, bacterial translocation and inflammatory reaction as well as improved 7-day survival. These effects were seen both in a pretreatment and in a treatment setting. The fact that this product is readily available and safe should encourage further clinical investigations in settings of intestinal IRI, organ preservation and transplantation.

INT-767-A Dual Farnesoid-X Receptor (FXR) and Takeda G Protein-Coupled Receptor-5 (TGR5) Agonist Improves Survival in Rats and Attenuates Intestinal Ischemia Reperfusion Injury.

Intestinal ischemia is a potentially catastrophic emergency, with a high rate of morbidity and mortality. Currently, no specific pharmacological treatments are available. Previous work demonstrated that pre-treatment with obeticholic acid (OCA) protected against ischemia reperfusion injury (IRI). Recently, a more potent and water-soluble version has been synthesized: Intercept 767 (INT-767). The aim of this study was to investigate if intravenous treatment with INT-767 can improve outcomes after IRI. In a validated rat model of IRI (60 min ischemia + 60 min reperfusion), three groups were investigated (n = 6/group): (i) sham: surgery without ischemia; (ii) IRI + vehicle; and (iii) IRI + INT-767. The vehicle (0.9% NaCl) or INT-767 (10 mg/kg) were administered intravenously 15 min after start of ischemia. Endpoints were 7-day survival, serum injury markers (L-lactate and I-FABP), histology (Park-Chiu and villus length), permeability (transepithelial electrical resistance and endotoxin translocation), and cytokine expression. Untreated, IRI was uniformly lethal by provoking severe inflammation and structural damage, leading to translocation and sepsis. INT-767 treatment significantly improved survival by reducing inflammation and preserving intestinal structural integrity. This study demonstrates that treatment with INT-767 15 min after onset of intestinal ischemia significantly decreases IRI and improves survival. The ability to administer INT-767 intravenously greatly enhances its clinical potential.

Protective Effect of Oxygen and Isoflurane in Rodent Model of Intestinal Ischemia-Reperfusion Injury.

Animal research in intestinal ischemia-reperfusion injury (IRI) is mainly performed in rodent models. Previously, intraperitoneal (I.P.) injections with ketamine-xylazine mixtures were used. Nowadays, volatile anesthetics (isoflurane) are more common. However, the impact of the anesthetic method on intestinal IRI has not been investigated. We aim to analyze the different anesthetic methods and their influence on the extent of intestinal IRI in a rat model. Male Sprague-Dawley rats were used to investigate the effect of I.P. anesthesia on 60 min of intestinal ischemia and 60 min of reperfusion in comparison to hyperoxygenation (100% O2) and volatile isoflurane anesthesia. In comparison to I.P. anesthesia with room air (21% O2), supplying 100% O2 improved 7-day survival by cardiovascular stabilization, reducing lactic acidosis and preventing vascular leakage. However, this had no effect on the intestinal epithelial damage, permeability, and inflammatory response observed after intestinal IRI. In contrast to I.P. + 100% O2, isoflurane anesthesia reduced intestinal IRI by preventing ongoing low-flow reperfusion hypotension, limiting intestinal epithelial damage and permeability, and by having anti-inflammatory effects. When translating the aforementioned results of this study to clinical situations, such as intestinal ischemia or transplantation, the potential protective effects of hyperoxygenation and volatile anesthetics require further research.

Proton Pump Inhibitors Reduce Duodenal Eosinophilia, Mast Cells, and Permeability in Patients With Functional Dyspepsia.

BACKGROUND & AIMS: Despite the growing recognition of duodenal alterations in the pathophysiology of functional dyspepsia (FD), the effect and mechanism of proton pump inhibitors (PPIs) or first-line therapy remain unclear. We studied duodenal and systemic alterations in relation to PPI therapy in patients with FD and healthy volunteers (HVs). METHODS: We performed a prospective interventional study assessing symptoms (Patient Assessment of Gastrointestinal Symptom Severity Index), duodenal alterations, and systemic factors in patients with FD ("FD-starters") and HVs before and after PPI therapy (pantoprazole 40 mg once daily for 4 weeks). Duodenal mucosal eosinophils, mast cells and permeability were quantified. Luminal pH and bile salts were determined in duodenal aspirates. Procedures were also performed in PPI-refractory patients with FD ("FD-stoppers") before and 8 weeks after PPI withdrawal. Between- and within-group changes from baseline and associations with duodenal or systemic factors were analyzed using linear mixed models. RESULTS: The study was completed by 30 HV, 27 FD-starters, and 18 FD-stoppers. Symptoms and duodenal eosinophils, mast cells (all, P < .0001), and paracellular passage (P = .02) were significantly higher in FD-starters vs HVs and reduced with PPI therapy. Symptoms and duodenal immune cells also decreased in FD-stoppers off PPIs. In contrast, immune cells and permeability increased in HVs on PPIs. Dyspeptic symptoms correlated with eosinophils before and during PPI therapy, and increased eosinophils and permeability in HVs on PPIs were associated with changes in bile salts. CONCLUSIONS: We provide the first prospective evidence for eosinophil-reducing effects as a therapeutic mechanism of PPIs in FD, with differential effects in HVs pointing to a role of luminal changes. ClinicalTrials.gov, Number: NCT03545243.

Duodenal Dysbiosis and Relation to the Efficacy of Proton Pump Inhibitors in Functional Dyspepsia.

Proton pump inhibitors (PPI) may improve symptoms in functional dyspepsia (FD) through duodenal eosinophil-reducing effects. However, the contribution of the microbiome to FD symptoms and its interaction with PPI remains elusive. Aseptic duodenal brushings and biopsies were performed before and after PPI intake (4 weeks Pantoprazole 40 mg daily, FD-starters and controls) or withdrawal (2 months, FD-stoppers) for 16S-rRNA sequencing. Between- and within-group changes in genera or diversity and associations with symptoms or duodenal factors were analyzed. In total, 30 controls, 28 FD-starters and 19 FD-stoppers were followed. Mucus-associated Porphyromonas was lower in FD-starters vs. controls and correlated with symptoms in FD and duodenal eosinophils in both groups, while Streptococcus correlated with eosinophils in controls. Although clinical and eosinophil-reducing effects of PPI therapy were unrelated to microbiota changes in FD-starters, increased Streptococcus was associated with duodenal PPI effects in controls and remained higher despite withdrawal of long-term PPI therapy in FD-stoppers. Thus, duodenal microbiome analysis demonstrated differential mucus-associated genera, with a potential role of Porphyromonas in FD pathophysiology. While beneficial effects of short-term PPI therapy were not associated with microbial changes in FD-starters, increased Streptococcus and its association with PPIeffects in controls suggest a role for duodenal dysbiosis after long-term PPI therapy.

Methadone Reverses Analgesic Tolerance Induced by Morphine Pretreatment.

BACKGROUND: Opiates such as morphine are the most powerful analgesics, but their protracted use is restrained by the development of tolerance to analgesic effects. Recent works suggest that tolerance to morphine might be due to its inability to promote mu opioid receptor endocytosis, and the co-injection of morphine with a mu opioid receptor internalizing agonist like [D-Ala(2),N-Me-Phe(4),Gly-ol(5)]enkephalin reduces tolerance to morphine. So far, no studies have been conducted to evaluate the ability of methadone to reduce morphine tolerance in morphine-pretreated animals, a treatment sequence that could be encountered in opiate rotation protocol. We investigated the ability of methadone (a mu opioid receptor internalizing agonist used in therapy) to reverse morphine tolerance and the associated cellular mechanisms in the periaqueductal gray matter, a region involved in pain control. METHODS: We measured analgesic response following a challenge dose of morphine in the hot plate test and investigated regulation of mu opioid receptor (coupling and endocytosis) and some cellular mechanisms involved in tolerance such as adenylate cyclase superactivation and changes in N-methyl-d-aspartate receptor subunits expression and phosphorylation state. RESULTS: A chronic treatment with morphine promoted tolerance to its analgesic effects and was associated with a lack of mu opioid receptor endocytosis, adenylate cyclase overshoot, NR2A and NR2B downregulation, and phosphorylation of NR1. We reported that a methadone treatment in morphine-treated mice reversed morphine tolerance to analgesia by promoting mu opioid receptor endocytosis and blocking cellular mechanisms of tolerance. CONCLUSIONS: Our data might lead to rational strategies to tackle opiate tolerance in the frame of opiate rotation.

A multicenter randomized controlled feasibility trial of a digital self-management intervention for adults with epilepsy.

OBJECTIVE: Self-management interventions may enhance health-related quality of life (HRQoL) in epilepsy. However, several barriers often impair their implementation in the real world. Digital interventions may help to overcome some of these barriers. Considering this, the Helpilepsy Plus Prototype was developed as a prototype smartphone-delivered self-care treatment program for adults with epilepsy. METHODS: The 12-week Helpilepsy Plus Prototype was evaluated through a randomized controlled feasibility trial with a waiting-list control (WLC) group. Outcome measurement at baseline and at 12 weeks assessed adherence to the prototype intervention and changes in epilepsy-related outcomes. The primary endpoint was patient autonomy measured with EASE, and secondary endpoints included HRQoL measured with QOLIE-31, health literacy measured with HLQ, anxiety, and depression symptoms measured with HADS. Semi-structured interviews were conducted with a heterogeneous sample of participants to assess user-friendliness and usefulness. The prototype program was delivered through the Neuroventis Platform (Neuroventis, BV, Overijse, Belgium), a certified medical device (under EU/MDD Class I, and EU/MDR grace period). RESULTS: Ninety-two patients were included (46 in the intervention group, 46 in WLC). Most participants (63%, 58/92 women, median age 30 years) had pharmacoresistant epilepsy (61%, 56/92). Only 22% of participants (10/46) in the intervention group completed at least half of all intervention sessions. No significant differences between the intervention group and WLC were observed. Although there was a larger proportion of patients in the intervention group with meaningful improvements in HRQoL compared to WLC (19/46 versus 11/46), the difference was not significant (p = 0.119). Qualitative feedback showed that participants would appreciate more personalization, such as adaptation of the content to their current epilepsy knowledge level, a more interactive interface, shorter text sections, and interaction through reminders and notifications. SIGNIFICANCE: Digital interventions should allow sufficient scope for personalization and interaction to increase patient engagement and enable benefits from self-care apps. Feedback loops allow the participatory development of tailored interventions. PLAIN LANGUAGE SUMMARY: In this study, we investigated the effectiveness of an app-based self-help intervention. Study participants were either randomly assigned to a group that had access to the app or a group that received access to the app after the end of the study. Although a larger proportion of participants in the intervention group showed a relevant improvement in quality of life, the difference between the two groups was not statistically significant. Less than one-fifth of participants in the intervention group attended at least half of all intervention sessions; patient feedback showed that patients required more personalization and interactive options.

Estrogens Play a Critical Role in Stress-Related Gastrointestinal Dysfunction in a Spontaneous Model of Disorders of Gut-Brain Interaction.

BACKGROUND: Disorders of the gut-brain interaction (DGBI), such as irritable bowel syndrome and functional dyspepsia, are more prevalent in women than in men, with a ratio of 2:1. Furthermore, stressful life events have been reported as one of the triggers for symptoms in DGBI patients. METHODS: Here, we studied the effect of an early-life stressor (maternal separation (MS)) on jejunal and colonic alterations, including colonic sensitivity and immune cells infiltration and activation in a validated spontaneous model of DGBI (BBDP-N), and investigated the involvement of ß-estradiol on stress-worsened intestinal alterations. RESULTS: We found that maternal separation exacerbated colonic sensitivity and mast cell and eosinophil infiltration and activation in females only. Ovariectomy partially rescued the stress phenotype by decreasing colonic sensitivity, which was restored by ß-estradiol injections and did not impact immune cells infiltration and activation. Stressed males exposed to ß-estradiol demonstrated similar intestinal alterations as MS females. CONCLUSION: Estrogen plays a direct critical role in colonic hypersensitivity in a spontaneous animal model of DGBI, while for immune activation, estrogen seems to be involved in the first step of their recruitment and activation. Our data point towards a complex interaction between stress and ß-estradiol in DGBI.

Lactobacillus rhamnosus CNCM I-3690 decreases subjective academic stress in healthy adults: a randomized placebo-controlled trial.

Psychological stress negatively affects the intestinal barrier function in animals and humans. We aimed to study the effect of Lactobacillus rhamnosus CNCM I-3690 on intestinal permeability and stress-markers during public speech. Healthy students were randomized to L. rhamnosus-containing (test) or acidified (placebo) milk consumed twice daily for 4 weeks, with 46 subjects per treatment group. Small intestinal permeability was quantified by a 2 h urinary lactulose-mannitol ratio (LMR, primary outcome), fractional excretion of lactulose (FEL) and mannitol (FEM). Salivary cortisol, State-Trait Anxiety Inventory (STAI) and Perceived Stress scores (PSS) were collected. No between-treatment differences were found for LMR (p = .71), FEL or FEM. Within-treatment analyses showed similar LMR and FEL but a stress-induced increase of FEM with the placebo (p < .05) but not test product. Despite a similar increase in salivary cortisol, the stress-induced increase in STAI was significantly lower with the test product vs. placebo (p = .01). Moreover, a stress-preventative effect of the probiotic was found for PSS and more pronounced in subjects with high stress-induced cortisol (p = .01). While increased FEM was mediated by salivary cortisol levels, the effect of the test product on subjective stress was not mediated by changes in FEM. No serious adverse events occurred. In conclusion, we demonstrated that L. rhamnosus CNCM I-3690 prevented stress-induced hyperpermeability to mannitol. Subjective but not objective stress-markers were reduced with L. rhamnosus vs. placebo, suggesting anxiolytic effects, which were independent of barrier stabilization and attractive for the reduction of stress in both health and disease. Clinicaltrials.gov, number NCT03408691.

Animal Models for Functional Gastrointestinal Disorders.

Functional gastrointestinal disorders (FGID), such as functional dyspepsia (FD) and irritable bowel syndrome (IBS) are characterized by chronic abdominal symptoms in the absence of an organic, metabolic or systemic cause that readily explains these complaints. Their pathophysiology is still not fully elucidated and animal models have been of great value to improve the understanding of the complex biological mechanisms. Over the last decades, many animal models have been developed to further unravel FGID pathophysiology and test drug efficacy. In the first part of this review, we focus on stress-related models, starting with the different perinatal stress models, including the stress of the dam, followed by a discussion on neonatal stress such as the maternal separation model. We also describe the most commonly used stress models in adult animals which brought valuable insights on the brain-gut axis in stress-related disorders. In the second part, we focus more on models studying peripheral, i.e., gastrointestinal, mechanisms, either induced by an infection or another inflammatory trigger. In this section, we also introduce more recent models developed around food-related metabolic disorders or food hypersensitivity and allergy. Finally, we introduce models mimicking FGID as a secondary effect of medical interventions and spontaneous models sharing characteristics of GI and anxiety-related disorders. The latter are powerful models for brain-gut axis dysfunction and bring new insights about FGID and their comorbidities such as anxiety and depression.

Extracellular vesicles derived from mesenchymal stromal cells mitigate intestinal toxicity in a mouse model of acute radiation syndrome.

BACKGROUND: Human exposure to high doses of radiation resulting in acute radiation syndrome and death can rapidly escalate to a mass casualty catastrophe in the event of nuclear accidents or terrorism. The primary reason is that there is presently no effective treatment option, especially for radiation-induced gastrointestinal syndrome. This syndrome results from disruption of mucosal barrier integrity leading to severe dehydration, blood loss, and sepsis. In this study, we tested whether extracellular vesicles derived from mesenchymal stromal cells (MSC) could reduce radiation-related mucosal barrier damage and reduce radiation-induced animal mortality. METHODS: Human MSC-derived extracellular vesicles were intravenously administered to NUDE mice, 3, 24, and 48 h after lethal whole-body irradiation (10 Gy). Integrity of the small intestine epithelial barrier was assessed by morphologic analysis, immunostaining for tight junction protein (claudin-3), and in vivo permeability to 4 kDa FITC-labeled dextran. Renewal of the small intestinal epithelium was determined by quantifying epithelial cell apoptosis (TUNEL staining) and proliferation (Ki67 immunostaining). Statistical analyses were performed using one-way ANOVA followed by a Tukey test. Statistical analyses of mouse survival were performed using Kaplan-Meier and Cox methods. RESULTS: We demonstrated that MSC-derived extracellular vesicle treatment reduced by 85% the instantaneous mortality risk in mice subjected to 10 Gy whole-body irradiation and so increased their survival time. This effect could be attributed to the efficacy of MSC-derived extracellular vesicles in reducing mucosal barrier disruption. We showed that the MSC-derived extracellular vesicles improved the renewal of the small intestinal epithelium by stimulating proliferation and inhibiting apoptosis of the epithelial crypt cells. The MSC-derived extracellular vesicles also reduced radiation-induced mucosal permeability as evidenced by the preservation of claudin-3 immunostaining at the tight junctions of the epithelium. CONCLUSIONS: MSC-derived extracellular vesicles promote epithelial repair and regeneration and preserve structural integrity of the intestinal epithelium in mice exposed to radiation-induced gastrointestinal toxicity. Our results suggest that the administration of MSC-derived extracellular vesicles could be an effective therapy for limiting acute radiation syndrome.

Duodenal acidification induces gastric relaxation and alters epithelial barrier function by a mast cell independent mechanism.

Duodenal hyperpermeability and low-grade inflammation in functional dyspepsia is potentially related to duodenal acid exposure. We aimed to evaluate in healthy volunteers the involvement of mast cell activation on the duodenogastric reflex and epithelial integrity during duodenal acidification. This study consisted of 2 parts: (1) Duodenal infusion of acid or saline during thirty minutes in a randomized, double-blind cross-over manner with measurement of intragastric pressure (IGP) using high resolution manometry and collection of duodenal biopsies to measure epithelial barrier function and the expression of cell-to-cell adhesion proteins. Mast cells and eosinophils were counted and activation and degranulation status were assessed. (2) Oral treatment with placebo or mast cell stabilizer disodiumcromoglycate (DSCG) prior to duodenal perfusion with acid, followed by the procedures described above. Compared with saline, acidification resulted in lower IGP (P < 0.01), increased duodenal permeability (P < 0.01) and lower protein expression of claudin-3 (P < 0.001). Protein expression of tryptase (P < 0.001) was increased after acid perfusion. Nevertheless, an ultrastructural examination did not reveal degranulation of mast cells. DSCG did not modify the drop in IGP and barrier dysfunction induced by acid. Duodenal acidification activates an inhibitory duodenogastric motor reflex and, impairs epithelial integrity in healthy volunteers. However, these acid mediated effects occur independently from mast cell activation.

Colonic hypersensitivity and low-grade inflammation in a spontaneous animal model for functional gastrointestinal disorders.

BACKGROUND: A complex interplay between a failing intestinal barrier and low-grade inflammation leading to sensorimotor disturbances is an often-cited mechanism in the pathogenesis of functional gastrointestinal disorders (FGID). However, the cause-consequence relationship between these features has not been clearly established. We previously described jejunal alterations in the normoglycemic BB-rat (BBDP-N) model proposing this model as a suitable animal model to study FGID pathophysiology. The current study explores colonic permeability, inflammation, and sensitivity of the BB-rat. METHODS: Colonic tissue of BBDP-N and control (BBDR) rats at 50, 90, 110, 160, and 220 days (n ≥ 7 per group) was used to assess intestinal permeability in Ussing chambers and inflammation, including infiltration by eosinophils, mast cells, and eosinophil peroxidase (EPO) activity. Anxiety-like symptoms were evaluated at 50, 90, and 220 days and colonic sensitivity at 160 and 220 days by measuring the visceromotor response (VMR) to isobaric colorectal distensions. KEYS RESULTS: Lamina propria eosinophil and mast cell infiltration and increased EPO activity were demonstrated from 90 days onward. Increased permeability and myenteric ganglionitis were observed in the oldest BBDP-N rats. At 220 days, the VMR was significantly increased suggesting colonic hypersensitivity. At the same age, increased anxiety-like behavior was observed. CONCLUSION AND INFERENCES: We demonstrated a lamina propria eosinophil and mast cell infiltration preceding visceral hypersensitivity in the colon of the BBDP-N rat, reminiscent of patients with FGID. These findings help elucidating pathogenetic pathways in FGID and further validate the BBDP-N rat as an attractive model to study pathophysiology and therapy of FGID.