<p>The small intestine holds an intrinsic ability to digest and absorb nutrients from the food we intake without intervention from the central nervous system. This ability is made possible by the population of cells that inhabit the gut, particularly interstitial cells of Cajal of the myenteric plexus and sensory primary intrinsic neurons (AH cells), which ultimately influence muscle function and motility. The AH cells are the first neurons in the hierarchy of sensory neurons in the gut and are therefore a perfect candidate to test the effects of <em>Bifidobacterium longum</em> NCC3001 supernatant since in a physiological setting the metabolites secreted by this bacterium can interact with the AH cells directly or indirectly through absorption by the mucosa.</p> <p>The probiotic <em>Bifidobacterium</em> <em>longum</em> NCC3001 has been shown to normalize anxiety-like behaviour and hippocampal brain derived neurotropic factor (BDNF) levels in mice infected with <em>Trichuris</em> <em>muris </em>in a model of infectious colitis. Utilizing a chronic model of colitis, a study was conducted to decipher whether or not the anxiolytic effects of <em>Bifidobacterium longum</em> NCC3001 involved the vagus. My specific objective in this study was to find evidence for interaction between <em>B.longum</em> NCC3001 and myenteric neurons as a potential route for <em>B.longum</em> NCC3001 to influence CNS function. We assessed a cell’s electro-responsiveness through spike discharge, which is the number of action potentials elicited in response to a supra-threshold depolarizing current injection.</p> <p>The electro-responsiveness of neurons perfused with <em>B. longum</em> NCC3001 supernatant (conditioned medium; n = 4) was significantly reduced compared to the control group (those perfused with Krebs solution; n = 5; <em>P</em> = 0.016). The electro-responsiveness of neurons perfused with the conditioned medium was also significantly lower than that of neurons perfused with unconditioned group (MRS growth medium alone) group (n = 4; <em>P</em> = 0.029). In comparing the excitabilities of the neurons in the control group with that of the control media group, there was no statistical difference (<em>P</em> = 0.29).</p> <p>In subsequent studies, the objective was to identify the AH cells and to determine the effect of <em>B. longum</em> NCC3001 conditioned medium on this population of cells. The electro-responsiveness as measured through spike discharge of AH cells perfused with the conditioned medium (n = 5) was significantly reduced compared to neurons perfused with the unconditioned medium (n = 5; <em>P</em> = 0.02). Sensory neurons perfused with the conditioned medium (n = 9) exhibited a significant reduction in their instantaneous input resistances compared to neurons perfused with the unconditioned medium (n = 8; <em>P </em>= 0.01). There was also a significant reduction in the time-dependent input resistance of neurons perfused with the conditioned medium (n = 9) compared to neurons perfused with the unconditioned medium (n = 8; <em>P </em>= 0.02). In addition, perfusion of the conditioned medium over sensory neurons (n = 9) significantly reduced the magnitude of the hyperpolarization-activated cationic current (<em>I</em>_h) compared to neurons perfused with the unconditioned medium (n = 8; <em>P</em> = 0.0003). Furthermore, there was also a significant reduction in the action potential half width duration of myenteric sensory neurons perfused with conditioned medium (n = 5) compared to that exhibited by neurons perfused with the unconditioned medium (n = 5; <em>P</em> = 0.008).</p> <p>In later experiments, we wanted to gain a more comprehensive understanding of the effect of this bacterium on the gut so we evaluated its effects on the gut musculature. Upon full immersion, the supernatant of <em>Bifidobacterium longum</em> NCC3001 (conditioned medium) caused an initial depolarization of the circular smooth muscle cell. This depolarization continued until the slow wave oscillations in these cells ceased and membrane potential would plateau. Several minutes after this plateau, the slow wave oscillations reappeared and the cell was significantly hyperpolarized relative to the conditions before conditioned medium was added. The resting membrane potential of circular smooth muscle cells in Krebs solution was -54.3 mV and -70.3 mV approximately two minutes after full immersion by the supernatant when the cell was hyperpolarized and a stable recorded was achieved (n = 7; <em>P</em> = 0.02). The average time of onset of depolarization was 18.6 s and the average change in membrane potential (depolarization) from onset of effect to its plateau was 14.0 mV (n = 7). Occasionally, the addition of the conditioned medium only caused an immediate but slight depolarization (n = 3) and in other cases caused only a hyperpolarization of the cell (n = 3) with no significant changes in any slow wave characteristics in either case. Furthermore, any cells that exhibited the waxing and waning of the slow wave lost this pattern upon the addition of the conditioned medium (n = 10).</p> <p>In attempts to understand the role of neurotransmission in this system, we conducted several experiments whereby carbachol (acetylcholine agonist) and L-NNA (nitric oxide synthase inhibitor) were administered to the muscle. Prior to the addition of 1μM carbachol or 2e^-4M L-NNA, we would only observe the pacemaker slow wave associated with the interstitial cells of Cajal of the myenteric plexus during the perfusion of Krebs solution. Upon the addition of carbachol (n = 3) or L-NNA (n = 4), we would observe a second slower frequency pattern appear, referred to as a waxing and waning pattern.</p> / Master of Science (MSc)

<p>The organs of the gastrointestinal (GI) tract display a variety of motor patterns, involved in grinding, mixing, enhancing absorption and propulsion of nutrients and waste products. Specialized motor patterns are generated by unique mechanisms inherent to the GI segment in which they are found. Rhythmic contractions are a feature of most motor patterns. Slow wave driven peristalsis is an acknowledged motor pattern associated with interstitial cells of Cajal (ICC) pacemakers, but propulsive motor patterns which are blocked by tetrodotoxin are seen to be exclusively generated by the enteric nervous system (ENS). This has not been proven, however, and the origin of rhythmicity of propulsive motor patterns needs further study, particularly related to a potential role of the pacemaker ICC found throughout the GI tract. The aim of this study was exploring the mechanisms which underlie various GI motor patterns, with particular focus on the origin of rhythmicity of these patterns.</p> <p>I have demonstrated with manometry and spatiotemporal maps that murine rhythmic propulsion requires a myogenic pacemaker which is evoked by acetylcholine and substance P; nitric oxide is not involved. Calcium imaging evidence suggests that the pacemaker is the ICC of the deep muscular plexus, as these cells rhythmically activate to substance P. I observed rhythmic contractility patterns in human antrum, pylorus and duodenum when stimulated with carbachol. The hypothesis emerged that the ENS modifies the pyloric pacemaker into unique rhythmic patterns. Colonic muscle strip contractility from the rat has a low frequency rhythmic pattern which is myogenic. This pattern is augmented by the conditioned media from the probiotic <em>E. coli</em> <em>Nissle 1917</em> through a non-neural mechanism.</p> <p>The current explanation of entirely ENS generated motor patterns is not accurate. The ENS plays an important role in stimulating and regulating GI motor patterns in conjunction with myogenic pacemakers. It is only through acknowledgment of all GI cell types that we can understand the mechanisms governing motility.</p> / Master of Science (MSc)

<p>Stress is known to play an important role in the natural history of gastrointestinal diseases, and functional disorders in particular. In health, activation of the stress response serves to maintain homeostasis in response to harmful stimuli. However, prolonged activation of the stress response can become maladaptive and contribute to the initiation and maintenance of symptoms in disorders such as irritable bowel syndrome (IBS). The mechanisms underlying this detrimental effect are unclear. This thesis investigates this relationship by examining the influence of 10 days of water avoidance stress on a murine model of acute bacterial gastroenteritis; a known trigger in a subset of IBS patients. Results indicate that stress can increase the level of the stress hormone norepinephrine in the gut. However, the overall influence of host stress during infection proves to be beneficial in this model, with decreased colonic inflammation and earlier clearance of the pathogen. Next, we utilized the olfactory bulbectomy (OBx) model of depression comorbid anxiety, which shows a heightened stress response, to examine mechanism underlying stress-mediated susceptibility in a more chronic setting. OBx resulted in increased neural activity and motility in the gut, and a change in composition of gut microbiota. These responses were not accompanied by changes in gut permeability or immune activation. Thus stress alters the habitat of commensal bacteria via a neurally mediated change in colonic motility. These results have bearing on the ability of stress to alter the microbiota: a feature of functional GI disorders.</p> / Doctor of Philosophy (Medical Science)

<p>Emerging evidence indicates that the commensal microbiota communicates with the brain and influences behavior. In animal models, perturbation of the microbiota is accompanied by changes in brain-derived neurotrophic factor (BDNF) levels in the brain. However, underlying mechanisms are unknown. We investigated whether vagal-parasympathetic and sympathetic branches of the autonomic nervous system are involved in the microbiota-gut-brain signalling and attempt to identify specific brain regions that are responsive to alterations in the intestinal microbiota. Specific pathogen-free Balb/c mice, with or without surgical vagotomy or chemical sympathectomy, received oral non-absorbable antimicrobials (ATM) <em>ad libitum</em> for 7 days. Behavior was tested on day 7 in the light/dark preference and step-down latency tests. Specific brain regions were sectioned and stained for the neuronal activation marker, <em>c-fos</em>. Perturbation of the microbiota significantly enhanced the exploratory behavior of mice in both tests and increased the expression of <em>c-fos</em> and phosphorylated <em>c-fos</em> in the hippocampus and dentate gyrus. <em>c-fos</em> expression in the nucleus of the solitary tract was unaffected and neither vagal-parasympathetic nor sympathetic neurotransmission were required for induction of the behavioral change following perturbation of the microbiota. Instability of the commensal microbiota enhances the activation of the hippocampal formation and influences host behavior in a manner that is independent of vagal-parasympathetic and sympathetic autonomic neurotransmission.</p> / Master of Health Sciences (MSc)

<p>Inflammatory Bowel Disease (IBD) is a chronic intestinal inflammation that has caused many challenges for healthcare providers in treating the disease and also altered the quality of life of the patients. The cure for IBD is still symptomatic-based; the causes mechanism and pathogenesis of IBD are to be further investigated. Currently, IBD has been considered as an excessive immune response to commensal flora that in normal condition is tolerable to the host. Antigen presenting cells (APCs) play an important role in the pathogenesis of IBD. Macrophage is one of the professional APCs that present antigen information to T cells and induce the T cell subtype proliferation. Aside from this role, macrophages also phagocytose pathogens and clean cell debris in thebody.</p> <p>T cell immunoglobulin and mucin domain (TIM)-4 is a glycoprotein expressed on the surface of macrophage, which recognizes phosphatidylserine (PS) that is expressed mainly on the surface of the early apoptotic cell phospholipid membrane; the latter is a negatively charged molecule that can bind to the TIM-4 to enhance the phagocytosing activity. In IBD, the loss of intestinal epithelial cells (IECs) due to apoptosis is prominent in the site of inflammation especially in ulcerative colitis (UC).</p> <p>The aim of this study is to elucidate whether there is an increase of TIM-4 expression in colitis mice model after exposure to excessive number of apoptotic IECs and whether TIM-4 plays a role in the development of colitis in mice.</p> <p>The expression of TIM-4 is measured with several tests; including flow cytometry, immunohistochemistry, western blotting and real time RT-PCR. In the first step, we tried to see if there is a difference in the TIM-4 expression in colitis mice and ethanol control mice. After the association was established, we further observed the role of TIM-4 in the pathogenesis of IBD by injecting TIM-4+ macrophages into the mice prior to inducing a mild colitis in the mice and finally injected neutralizing anti TIM-4 antibodies to block the available TIM-4 receptors.</p> <p>We found that TIM-4 expression was higher in a colitis mouse model compared to the control. Also by injecting TIM-4+ macrophages into the mice, the frequency of intestinal T regulatory (Treg) cells was decreased significantly. Finally in the group treated with anti-TIM-4 neutralizing antibodies prior to colitis induction, the frequency of intestinal Treg cells increased significantly and the inflammation response was less severe than the colitis control group. This study revealed, for the first time in the world, that TIM-4 expression in the colon of colitis mice was significantly increased, which suppressed Tregs and promoted T effector cells.</p> / Master of Science in Medical Sciences (MSMS)

<p>At birth the gastrointestinal (GI) tract is rapidly colonized by microbial organisms which exhibit considerable fluctuations in composition across the first two years of life. During this period, the enteric nervous system (ENS) continues to undergo significant structural and functional changes. In the present study, we investigated whether exposure to intestinal microbiota influences the postnatal development of the ENS. We focused our investigations on dopaminergic neurons as they are among the latest populations of neurons to differentiate during enteric development. The myenteric plexus of specific pathogen-free (SPF) and germ-free (GF) mice were examined in whole-mount preparations of the small and large intestine at three time-points: postnatal day 1 (P1), P7, and P28. The density of dopaminergic neurons did not differ significantly between SPF and GF mice in any region of the intestine examined at P1. However, at P7, GF mice had significantly fewer myenteric dopaminergic neurons in the ileum than did SPF mice, and this difference was maintained at P28.</p> <p>The proportion of enteric dopaminergic neurons has been shown to be dependent upon the availability of serotonin. In the GI tract, serotonin can be of neuronal or enterochromaffin (EC) cell origin. We therefore tested the hypothesis that reductions in myenteric dopaminergic neuron densities in the ileum of GF mice were secondary to changes in enteric serotonergic neuron densities or EC cell frequencies. Neither serotonergic neurons nor EC cell numbers were affected by GF status during the postnatal period. The reduction in dopaminergic neurons seen in GF mice must therefore be attributable to a mechanism that has yet to be determined.</p> <p>These findings are consistent with the notion that enteric microbiota can influence the development of late-born neuronal populations. The reduced proportion of dopaminergic neurons in the ileum of GF mice at P7 and P28 may contribute to the previously described altered motility patterns in postnatal GF mice.</p> / Master of Science (MSc)

<p>Nonsteroidal anti-inflammatory drugs are among the most commonly used prescription and over-the-counter medications, but they often produce significant gastrointestinal ulceration and bleeding, particularly in elderly patients and patients with certain co-morbidities. Novel anti-inflammatory drugs are seldom tested in animal models that mimic the high-risk human users, leading to an underestimate of the true toxicity of these drugs. In the present study we examined the effects of two novel NSAIDs and two commonly used NSAIDs in models in which mucosal defence was expected to be impaired. Naproxen, celecoxib, ATB-346 (a hydrogen sulfide- and naproxen-releasing compound) and NCX 429 (a nitric oxide- and naproxen-releasing compound) were evaluated in healthy, arthritic, obese, hypertensive rats, and in rats of advanced age (19 months) and rats co-administered low-dose aspirin and/or omeprazole. In all models except hypertension, greater gastric and/or intestinal damage was observed when naproxen was administered in these models than in healthy rats. Celecoxib-induced damage was significantly increased when co-administered with low-dose aspirin and/or omeprazole. In contrast, ATB-346 and NCX 429, when tested at doses that were as effective as naproxen and celecoxib in reducing inflammation and inhibiting cyclooxygenase activity, did not produce significant gastric or intestinal damage in any of the models. These results demonstrate that animal models of human co-morbidities display the same increased susceptibility to NSAID-induced gastrointestinal damage as observed in humans. Moreover, two novel NSAIDs that release mediators of mucosal defence (hydrogen sulfide and nitric oxide) do not induce significant gastrointestinal damage in these models of impaired mucosal defence.</p> / Master of Science (MSc)

<p>Intestinal microbiota are key determinants of gut homeostasis and affect various gut physiological and immune processes. Co-evolution has enabled the host and intestinal microbes to exist in a mutualistic relationship. However, interactions between the host and its intestinal microbiota exist in a delicate balance between mutualism and pathogenicity. Maintenance or disruption of this balance depends on a complex interplay between the microbiota and the host, as well as other gut luminal factors, including diet, that are poorly understood. The main goal of this thesis has been to study the host-gut luminal interactions that regulate gut physiology and immunity. In particular, <strong>Chapter 2</strong> centers on investigating the effect of perturbing the intestinal barrier using a non-steroidal inflammatory drug on host-microbial and dietary interactions in a mouse model of gluten sensitivity. I demonstrated that indomethacin-induced increase in intestinal permeability is associated with altered intestinal microbiota composition, systemic antibody development against intestinal bacteria and a shift in immune responses to the dietary antigen, gluten. <strong>Chapter 3</strong> focuses on investigating whether modulation of the intestinal microbiota can affect the host’s susceptibility to intestinal injury. I used mice with defective intracellular bacterial receptor signaling because discrimination between commensals and pathogens is, in part, achieved by a family of receptors that recognize conserved bacterial components. I demonstrated that the microbiota with which these mice are colonized influences the expression of RegIII-γ, a type of antimicrobial peptide, and susceptibility to intestinal injury. To gain further insight on the effect of microbiota on antimicrobial peptides, in <strong>Chapter 4</strong> we conducted a combination of gnotobiotic and <em>in-vitro</em> experiments where we identified that specific components of the microbiota differentially regulate RegIII expression. Further examination showed that <em>MyD88 a</em>nd <em>Ticam1 </em>genes, which are signaling adaptor proteins of pattern recognition receptors, are essential regulators of microbial–induced RegIII expression by intestinal epithelial cells. Collectively, the work presented in this thesis provides novel insight on the bi-directional interaction between the host and the gut luminal content as well as of potential beneficial effects of microbiota-modulating strategies in maintaining homeostasis and preventing disease.</p> / Doctor of Philosophy (Medical Science)

Crohn’s disease (CD) affects about 780,000 people in the United States alone, and it is estimated that 6-15 per 100,000 persons will receive a diagnosis of this disease each year. There currently is no cure for Crohn’s disease, and available medical therapies simply serve to alleviate the inflammation. This does not help treat fibrostenosis that Crohn’s disease patients may develop, which can only be treated surgically. Finding alternatives to treat CD requires an understanding of mechanisms at the biochemical level. In this thesis, we attempted to gain a better understanding of certain pathways found to be active in Crohn’s disease-affected ileal smooth muscle cells. We found an upregulation of the ER stress pathway via expression of its surrogate, the GRP78 protein. We also showed evidence that the phosphoinositide 3-kinase (PI3K) pathway, a key proliferative pathway, is linked to ER stress in these cells, and is an upstream driving force of the ER stress response. Further research on the link between the PI3K and ER stress pathways needs to be conducted, and can potentially serve as a target for therapeutics to help reduce proliferation in fibrostenotic Crohn’s disease-affected ileal smooth muscle cells.

Isovaleric Acid (IVA) is a 5-carbon branched chain fatty acid present in fermented foods and produced by the fermentation of leucine by colonic bacteria. IVA activates G-protein coupled receptors such as FFAR2, FFAR3, and OR51E1 known to be expressed on enteric neurons and enteroendocrine cells. We previously reported that the shorter, straight chain fatty acids acetate, propionate and butyrate, differentially affect colonic propulsion; however, the effect of branched chain fatty acids on gastrointestinal motility is unknown. We hypothesize that IVA relaxes smooth muscle in a cAMP/PKA dependent manner by direct action on smooth muscle cells. IVA will also decrease peristalsis and encourage retention of luminal contents. This thesis investigates the effect of IVA on smooth muscle tension and peristaltic activity in isolated colon and individual smooth muscle cells. Colon segments from C57BL/6J mice were placed in a longitudinal orientation in organ baths in Krebs buffer and fastened to force transducers. Segments were contracted with 10 μM acetylcholine (ACh) and the effects of IVA at several concentrations were measured in the absence and presence of Nitric Oxide Synthase inhibitor L-N-nitroarginine (L-NNA), neuronal action potential inhibitor tetrodotoxin (TTX), and adenylate cyclase inhibitor SQ22536. To study individual live cells, mouse smooth muscle was isolated from colon, suspended in smooth muscle buffer, and after contraction with ACh were relaxed with micromolar concentrations of IVA. For peristalsis studies, whole colonic segments isolated from C57BL/6J were catheterized and placed horizontally in organ baths with circulating Krebs buffer. The colon was clamped on the anal end, and a solution (5 μL per mm of colon length) of either Krebs buffer or 50 mM IVA was delivered from the oral end to the lumen. Video of the peristalsis was then analyzed for diameter, changes in diameter, velocity of diameter changes along the length of the colon, normalized to the anatomical changes in the proximal region. IVA in concentrations of 10 mM to 50 mM relaxed the ACh-induced contraction in a sigmoidal fashion. In separate studies, L-NNA nor TTX affected the ability of IVA to inhibit relaxation. SQ22536 inhibited IVA induced relaxation in longitudinal colon compared to vehicle control. In isolated cells, SQ22536 and PKA inhibitor H-89 inhibited IVA-induced relaxation. In peristalsis studies, 50 mM IVA in Krebs buffer changed the character of the peristaltic action by increasing proximal diameter, inhibiting contractions in the proximal end of the colon, and decreasing overall velocity of peristaltic contractions in the proximal region. The data indicate that the branched chain fatty acid IVA causes a concentration-dependent relaxation of colonic smooth muscle that is direct to the smooth muscle and independent of neuronal activity. This relaxation is cAMP/PKA dependent. In addition to the direct relaxation of smooth muscle, intraluminal IVA decreased overall colonic propulsive activity and encouraged retention of the luminal contents. We conclude that the ingestion and production of branched chain fatty acids could affect overall GI motility and is an area for study in dietary and therapeutic control of bowel activity.