Jennifer Jury

The Intestinal Microbiota Affect Central Levels of Brain-Derived Neurotropic Factor and Behavior in Mice

BACKGROUND & AIMS Alterations in the microbial composition of the gastrointestinal tract (dysbiosis) are believed to contribute to inflammatory and functional bowel disorders and psychiatric comorbidities. We examined whether the intestinal microbiota affects behavior and brain biochemistry in mice. METHODS Specific pathogen-free (SPF) BALB/c mice, with or without subdiaphragmatic vagotomy or chemical sympathectomy, or germ-free BALB/c mice received a mixture of nonabsorbable antimicrobials (neomycin, bacitracin, and pimaricin) in their drinking water for 7 days. Germ-free BALB/c and NIH Swiss mice were colonized with microbiota from SPF NIH Swiss or BALB/c mice. Behavior was evaluated using step-down and light preference tests. Gastrointestinal microbiota were assessed using denaturing gradient gel electrophoresis and sequencing. Gut samples were analyzed by histologic, myeloperoxidase, and cytokine analyses; levels of serotonin, noradrenaline, dopamine, and brain-derived neurotropic factor (BDNF) were assessed by enzyme-linked immunosorbent assay. RESULTS Administration of oral antimicrobials to SPF mice transiently altered the composition of the microbiota and increased exploratory behavior and hippocampal expression of BDNF. These changes were independent of inflammatory activity, changes in levels of gastrointestinal neurotransmitters, and vagal or sympathetic integrity. Intraperitoneal administration of antimicrobials to SPF mice or oral administration to germ-free mice did not affect behavior. Colonization of germ-free BALB/c mice with microbiota from NIH Swiss mice increased exploratory behavior and hippocampal levels of BDNF, whereas colonization of germ-free NIH Swiss mice with BALB/c microbiota reduced exploratory behavior. CONCLUSIONS The intestinal microbiota influences brain chemistry and behavior independently of the autonomic nervous system, gastrointestinal-specific neurotransmitters, or inflammation. Intestinal dysbiosis might contribute to psychiatric disorders in patients with bowel disorders.

Probiotics prevent bacterial translocation and improve intestinal barrier function in rats following chronic psychological stress

Background and aim: Chronic psychological stress, including water avoidance stress (WAS), induces intestinal mucosal barrier dysfunction and impairs mucosal defences against luminal bacteria. The aim of this study was to determine the ability of a defined probiotic regimen to prevent WAS induced intestinal pathophysiology. Methods: Male rats were subjected to either WAS or sham stress for one hour per day for 10 consecutive days. Additional animals received seven days of Lactobacillus helveticus and L rhamnosus in the drinking water prior to stress and remained on these probiotics for the duration of the study. Rats were then sacrificed, intestinal segments assessed in Ussing chambers, and mesenteric lymph nodes cultured to determine bacterial translocation. Results: All animals remained healthy for the duration of the study. Chronic WAS induced excess ion secretion (elevated baseline short circuit current) and barrier dysfunction (increased conductance) in both the ileum and colon, associated with increased bacterial adhesion and penetration into surface epithelial cells. Approximately 70% of rats subjected to WAS had bacterial translocation to mesenteric lymph nodes while there was no bacterial translocation in controls. Probiotic pretreatment alone had no effect on intestinal barrier function. However, WAS induced increased ileal short circuit current was reduced with probiotics whereas there was no impact on altered conductance. Pretreatment of animals with probiotics also completely abrogated WAS induced bacterial adhesion and prevented translocation of bacteria to mesenteric lymph nodes. Conclusion: These findings indicate that probiotics can prevent chronic stress induced intestinal abnormalities and, thereby, exert beneficial effects in the intestinal tract.

Proton Pump Inhibitors Exacerbate NSAID-Induced Small Intestinal Injury by Inducing Dysbiosis

BACKGROUND & AIMS Proton pump inhibitors (PPIs) and nonsteroidal anti-inflammatory drugs (NSAIDs) are among the most commonly used classes of drugs, with the former frequently coprescribed to reduce gastroduodenal injury caused by the latter. However, suppression of gastric acid secretion by PPIs is unlikely to provide any protection against the damage caused by NSAIDs in the more distal small intestine. METHODS Rats were treated with antisecretory doses of omeprazole or lanzoprazole for 9 days, with concomitant treatment with anti-inflammatory doses of naproxen or celecoxib on the final 4 days. Small intestinal damage was blindly scored, and changes in hematocrit were measured. Changes in small intestinal microflora were evaluated by denaturing gradient gel electrophoresis and reverse-transcription polymerase chain reaction. RESULTS Both PPIs significantly exacerbated naproxen- and celecoxib-induced intestinal ulceration and bleeding in the rat. Omeprazole treatment did not result in mucosal injury or inflammation; however, there were marked shifts in numbers and types of enteric bacteria, including a significant reduction (∼80%) of jejunal Actinobacteria and Bifidobacteria spp. Restoration of small intestinal Actinobacteria numbers through administration of selected (Bifidobacteria enriched) commensal bacteria during treatment with omeprazole and naproxen prevented intestinal ulceration/bleeding. Colonization of germ-free mice with jejunal bacteria from PPI-treated rats increased the severity of NSAID-induced intestinal injury, as compared with mice colonized with bacteria from vehicle-treated rats. CONCLUSIONS PPIs exacerbate NSAID-induced intestinal damage at least in part because of significant shifts in enteric microbial populations. Prevention or reversal of this dysbiosis may be a viable option for reducing the incidence and severity of NSAID enteropathy.

Probiotic treatment of rat pups normalises corticosterone release and ameliorates colonic dysfunction induced by maternal separation

Background: We previously showed that neonatal maternal separation (MS) of rat pups causes immediate and long-term changes in intestinal physiology. Aim: To examine if administration of probiotics affects MS-induced gut dysfunction. Methods: MS pups were separated from the dam for 3 h/day from days 4 to 19; non-separated (NS) pups served as controls. Twice per day during the separation period, 108 probiotic organisms (two strains of Lactobacillus species) were administered to MS and NS pups; vehicle-treated pups received saline. Studies were conducted on day 20, when blood was collected for corticosterone measurement as an indication of hypothalamus–pituitary–adrenal (HPA) axis activity, and colonic function was studied in tissues mounted in Ussing chambers. Ion transport was indicated by baseline and stimulated short-circuit current (Isc); macromolecular permeability was measured by flux of horseradish peroxidase (HRP) across colonic tissues; and bacterial adherence/penetration into the mucosa was quantified by culturing tissues in selective media. Colonic function and host defence were also evaluated at day 60. Results: Isc and HRP flux were significantly higher in the colon of MS versus NS pups. There was increased adhesion/penetration of total bacteria in MS pups, but a significant reduction in Lactobacillus species. Probiotic administration ameliorated the MS-induced gut functional abnormalities and bacterial adhesion/penetration at both day 20 and 60, and reduced the elevated corticosterone levels at day 20. Conclusions: The results indicate that altered enteric flora are responsible for colonic pathophysiology. Probiotics improve gut dysfunction induced by MS, at least in part by normalisation of HPA axis activity.

Epithelia Under Metabolic Stress Perceive Commensal Bacteria as a Threat

The normal gut flora has been implicated in the pathophysiology of inflammatory bowel disease and there is increased interest in the role that stress can play in gut disease. The chemical stressor dinitrophenol (DNP, uncouples oxidative phosphorylation) was injected into the ileum of laparotomized rats and mitochondria structure, epithelial permeability, and inflammatory cell infiltrate were examined 6 and 24 hours later. Monolayers of human colonic epithelial cells (T84, HT-29) were treated with DNP +/- commensal Escherichia coli, followed by assessment of epithelial permeability, bacterial translocation, and chemokine (ie, interleukin-8) synthesis. Delivery of DNP into rat distal ileum resulted in disruption of epithelial mitochondria; similar changes were noted in mildly inflamed ileal resections from patients with Crohns disease. Also, DNP-treated ileum displayed increased gut permeability and immune cell recruitment. Subsequent studies revealed deceased barrier function, increased bacterial translocation, increased production of interleukin-8, and enhanced mobilization of the transcription factor AP-1 in the model epithelial cell lines exposed to commensal bacteria (E. coli strains HB101 or C25), but only when the monolayers were pretreated with DNP (0.1 mmol/L). These data suggest that enteric epithelia under metabolic stress perceive a normally innocuous bacterium as threatening, resulting in loss of barrier function, increased penetration of bacteria into the mucosa, and increased chemokine synthesis. Such responses could precipitate an inflammatory episode and contribute to existing enteric inflammatory disorders.

Neonatal Maternal Separation Causes Colonic Dysfunction in Rat Pups Including Impaired Host Resistance

Previous studies have shown that early life stress in the form of intermittent maternal separation (MS) predisposes adult rats to develop stress-induced intestinal mucosal dysfunction and visceral hypersensitivity. However, the mechanism involved in the functional abnormalities is unclear. Our aim was to study immature animals during or shortly after exposure to MS to determine whether there are early pathophysiological changes in the gut. Sprague-Dawley rat pups were individually separated from the dam for 3 h/d from 4 to 21 d of age; nonseparated (NS) control pups remained in the home cage with the dam. On d 19–20, d 24–25, and d 29–30, blood was collected for corticosterone measurement, and colonic tissues were removed for functional and morphologic assessment. Corticosteroid levels were elevated in MS pups compared with NS, indicating that MS was indeed stressful. The distal colon demonstrated significantly enhanced ion secretion and macromolecular permeability at d 19–20 and d 24–25, returning to normal by d 29–30. Electron microscopy and bacterial culture studies indicated bacteria adhering to and penetrating into the colonic epithelium of the MS pups at all time points, while such events were rare in NS pups. The pathophysiological changes were inhibited by injecting pups sc with a corticotropin-releasing hormone (CRH) receptor antagonist daily during MS. Our studies indicate that early psychological trauma predisposes neonatal rats to develop persistent mucosal barrier dysfunction, including impaired host defense to luminal bacteria, by a mechanism involving peripheral CRH receptors.

Sites and mechanisms of action of neuropeptides on canine gastric motility differ in vivo and in vitro

Motilin, pentagastrin and substance P (SP), injected intra-arterially into the canine gastric corpus in vivo increased the amplitude of contractions by an action dependent on activation of cholinergic nerves; i.e. atropine or tetrodotoxin (TTX) completely blocked the responses to motilin and pentagastrin and increased the ED50 of SP. TTX and atropine were not equally effective in increasing the ED50 for SP in vivo and the effect of combining them depended on the order of their addition. Both were much more effective than the SP analog D-Pro2, D-Trp7,9 SP (DSP) which appeared to be a weak antagonist of actions dependent on neural activity. In strips from the same region in vitro no receptors dependent on cholinergic nerve activation could be demonstrated for any peptide; i.e., all were atropine- and TTX-insensitive. Motilin, as expected in the absence of such receptors caused no contractile response in vitro. SP, also as predicted, caused contractions suggesting that a smooth muscle receptor, independent of nerve activation was present. However contrary to expectation pentagastrin induced an atropine and TTX-insensitive increase in the amplitude and frequency of contractions. These results show that 1) the most sensitive sites of action of a number of excitatory peptides depend on cholinergic nerve function in vivo; 2) such sites or the nerve activity on which they depend cannot be demonstrated in vitro; 3) SP has an additional site of action on smooth muscle demonstrable in vivo and in vitro, but motilin does not; 4) pentagastrin has only an action dependent on nerve function in vivo, but manifests an action independent of nerve function in vitro. We conclude that sites and mechanisms of action of peptides cannot be assumed to be identical in vivo and in vitro. Actions dependent on nerves are often lost in vitro and not all smooth muscle actions can be demonstrated in vivo.

Commensal and probiotic bacteria influence intestinal barrier function and susceptibility to colitis in Nod1-/-; Nod2-/- mice.

Background: The intestinal microbiota regulates key host functions. It is unknown whether modulation of the microbiota can affect a genetically determined host phenotype. Polymorphisms in the Nucleotide oligomerization domain (Nod)‐like receptor family confer genetic risk for inflammatory bowel disease (IBD). We investigated whether the intestinal microbiota and the probiotic strain Bifidobacterium breve NCC2950 affect intestinal barrier function and responses to intestinal injury in Nod1−/−;Nod2−/− mice. Methods: Specific pathogen‐free (SPF) Nod1−/−;Nod2−/− mice and mice gnotobiotically derived with altered Schaedler flora (ASF) biota were used. SPF Nod1+/−;Nod2+/−littermates (generated by crossing SPF Nod1−/−;Nod2−/− and germ‐free C57BL/6 mice) and ASF Nod1+/−;Nod2+/− mice were used as controls. SPF mice were gavaged daily with 109‐CFU B. breve for 14 days before colitis induction. Denaturing gradient gel electrophoresis (DGGE) and real‐time polymerase chain reaction (PCR) were used to assess microbiota composition. Intestinal permeability was assessed by in vitro and in vivo techniques. Expressions of epithelial apical junction proteins, mucin, and antimicrobial proteins were assessed by quantitative reverse‐transcription PCR (qRT‐PCR) and immunofluorescence. Responses to intestinal injury were investigated using an acute experimental model of colitis. Results: Under SPF conditions, Nod1−/−;Nod2−/− mice had increased paracellular permeability, decreased E‐cadherin, and lower colonic antimicrobial RegIII‐&ggr; expression compared to Nod1+/−;Nod2+/− littermate controls. These changes were associated with increased susceptibility to colitis. ASF colonization or B. breve supplementation normalized RegIII‐&ggr; expression and decreased susceptibility to dextran sodium sulfate (DSS) colitis in Nod1−/−;Nod2−/− mice. Conclusions: The intestinal microbiota influences colitis severity in Nod1−/−;Nod2−/− mice. The results suggest that colonization strategies with defined commensals or exogenous specific probiotic therapy may prevent intestinal inflammation in a genetically predisposed host. (Inflamm Bowel Dis 2012)

Age-Associated Microbial Dysbiosis Promotes Intestinal Permeability, Systemic Inflammation, and Macrophage Dysfunction

Summary Levels of inflammatory mediators in circulation are known to increase with age, but the underlying cause of this age-associated inflammation is debated. We find that, when maintained under germ-free conditions, mice do not display an age-related increase in circulating pro-inflammatory cytokine levels. A higher proportion of germ-free mice live to 600 days than their conventional counterparts, and macrophages derived from aged germ-free mice maintain anti-microbial activity. Co-housing germ-free mice with old, but not young, conventionally raised mice increases pro-inflammatory cytokines in the blood. In tumor necrosis factor (TNF)-deficient mice, which are protected from age-associated inflammation, age-related microbiota changes are not observed. Furthermore, age-associated microbiota changes can be reversed by reducing TNF using anti-TNF therapy. These data suggest that aging-associated microbiota promote inflammation and that reversing these age-related microbiota changes represents a potential strategy for reducing age-associated inflammation and the accompanying morbidity.

Chronic peripheral administration of corticotropin-releasing factor causes colonic barrier dysfunction similar to psychological stress

Chronic psychological stress causes intestinal barrier dysfunction and impairs host defense mechanisms mediated by corticotrophin-releasing factor (CRF) and mast cells; however, the exact pathways involved are unclear. Here we investigated the effect of chronic CRF administration on colonic permeability and ion transport functions in rats and the role of mast cells in maintaining the abnormalities. CRF was delivered over 12 days via osmotic minipumps implanted subcutaneously in wild-type (+/+) and mast cell-deficient (Ws/Ws) rats. Colonic segments were excised for ex vivo functional studies in Ussing chambers [short-circuit current (Isc), conductance (G), and macromolecular permeability (horseradish peroxidase flux)], and analysis of morphological changes (mast cell numbers and bacterial host-interactions) was determined by light and electron microscopy. Chronic CRF treatment resulted in colonic mucosal dysfunction with increased Isc, G, and horseradish peroxidase flux in+/+but not in Ws/Ws rats. Furthermore, CRF administration caused mast cell hyperplasia and abnormal bacterial attachment and/or penetration into the mucosa only in+/+rats. Finally, selective CRF agonist/antagonist studies revealed that stimulation of CRF-R1 and CRF-R2 receptors induced the elevated secretory state and permeability dysfunction, respectively. Chronic CRF causes colonic barrier dysfunction in rats, which is mediated, at least in part, via mast cells. This information may be useful in designing novel treatment strategies for stress-related gastrointestinal disorders.