The characteristic loops of the small intestine arise during embryonic development from differential growth as the intestinal tube elongates against the constraint of its attached dorsal mesentery, which compresses the tube until it buckles into loops. The number and shape of loops are conserved for a given species and are predictable from tube and mesentery geometries and stiffnesses. Importantly, the mesentery readily accommodates a certain amount of stretch from the elongating tube before stiffening by several orders of magnitude and resisting further extension—thereby dictating how differential growth translates into buckling forces. While such constitutive nonlinearity is well appreciated in adult soft tissues, its determinants and consequences remain largely unexplored in buckling morphogenesis and embryogenesis as a whole. In this work, we undertake to establish a mechanistic link between molecular control of cell behaviors and organ-scale buckling morphogenesis of the small intestine. Using pharmacological treatments, mechanical testing, image analysis of tissue microstructure, and computational modeling, we test the hypothesis that actomyosin contractility regulates mesentery constitutive nonlinearity and thereby organ-scale buckling of the small intestine through its effects on extracellular matrix recruitment. Our findings suggest that highly contractile cells could act as a mechanical ‘clutch’, modulating the stiffening transition of the mesentery by compacting stiff matrix fibers that must be decompressed by applied forces before contributing to stretch resistance. However, we also find that low levels of contractility control the initial soft response of the mesentery through a mechanism largely independent of matrix fiber straightness and alignment. Despite the apparent simplicity of buckling from a mechanical standpoint, its underlying biological determinants are evidently quite complex. The present study begins to unpack those intricate links between the molecular and biophysical aspects of buckling morphogenesis by revealing how cell forces and matrix organization interactively dictate tissue-scale mechanics during development in sometimes counterintuitive ways.

Chick embryos derive nutrients from the yolk during incubation and transition to intestinal absorption posthatch. Nutrient uptake is mediated by digestive enzymes and membrane bound transporter proteins. The objective of this study was to determine expression profiles of nutrient transporters and digestive enzymes during incubation in the yolk sac membrane (YSM) and small intestine of Leghorn and Cobb chickens derived from 22–30 wk (young) and 45–50 wk (old) breeder flocks. Genes examined included peptide transporter PepT1, amino acid transporters EAAT3, CAT1, and B0AT, monosaccharide transporters SGLT1 and GLUT5, and digestive enzymes APN and SI. Expression of these genes was measured in YSM at embryonic day (e) 11, 13, 15, 17, 19, 20, and 21 and small intestine at e15, e17, e19, e20, and e21. Absolute quantification real-time PCR assessed gene expression. PepT1, APN, and B0AT expression in YSM peaked between e15 and e17 and then decreased until e21, while expression increased over time in the small intestine. SGLT1 and EAAT3 expression increased over time in the small intestine and YSM. There was minimal gene expression of SI in the YSM, while the small intestine had high expression. GLUT5 and CAT1 expression decreased in the YSM, while peaking at e19 then decreasing in the small intestine. Breed and flock age affected expression levels in some genes. These results demonstrate that these genes show tissue- and development-specific patterns of expression and that the YSM expresses many digestive enzymes and nutrient transporters associated with the small intestine. / Master of Science

Nutrient transporters in the small intestine are responsible for dietary nutrient assimilation and therefore the expression of these transporters can influence the overall nutrient status as well as the growth and development of the animal. This thesis examined correlated responses to selection in the developmental gene expression of the peptide transporter PepT1, the glutamate/aspartate transporter EAAT3, the sodium-dependent glucose transporter SGLT1, and the fructose transporter GLUT5 in the small intestine of chickens from lines divergently selected for high (HH) or low (LL) eight-week body weight and their reciprocal crosses, (HL and LH). Chicks were weighed and killed on embryonic day 20 (E20), day of hatch (DOH with no access to feed), and days 3 (D3), 7(D7), and 14 (D14) post hatch. Duodenum, jejunum, ileum and liver were collected. DNA extracted from liver was used to sex birds by PCR. RNA was extracted from the intestinal segments of four males and four females from each mating combination (MC) and time point except E20 HL males (n = 3) and D7 LL females (n = 2). Expression of nutrient transporters was assayed by real-time PCR using the relative quantification method. In comparing HH and LL males and females there was a line by segment interaction in PepT1 gene expression, with no segment difference in HH and greatest expression in the ileum of the LL (P < 0.05). There was also a MC by age by sex interaction for PepT1 gene expression (P < 0.0001) with peak gene expression occurring on DOH for LL females, on D7 for HH females, on D7 for LL males and D14 for HH males. Overall, females had greater EAAT3 expression (P < 0.03). Gene expression of EAAT3 was greatest in the ileum, intermediate in the jejunum, and least in the duodenum (P < 0.0007). There was an age by segment interaction for EAAT3 expression (P = 0.0002) and a MC by segment interaction (P < 0.02), with LL having greater expression than HH in the ileum. Females had greater SGLT1 expression than males (P < 0.0001). There was a sex by age interaction for the expression of SGLT1 (P < 0.0001). Females induced SGLT1 expression on DOH and maintained this level through D14, while males gradually increased expression through D7 and decreased expression by D14. These results indicate that expression of PepT1, EAAT3, SGLT1 are differentially expressed in male and female chickens regardless of selection for high or low juvenile body weight. These results also show a sexual dimorphism in the capacity to absorb peptides, anionic amino acids, and glucose from the intestine, which has implications for the poultry industry with regard to diet formulations for straight-run and sex-separate grow-out operations. In comparing male HH, HL, LH, and LL chicks, overall LL had the greatest level of expression (P <0.06), HH had the least level of expression (P < 0.006) and HL and LH had intermediate levels of expression (P < 0.06). Greatest PepT1 gene was expression in the ileum (P < 0.0003) and there was a MC by segment interaction with expression increasing from duodenum to ileum in LL, but there was no segment difference in any other MC (P < 0.08). Within each intestinal segment there was a MC difference (P < 0.02). There was an effect of sire for PepT1 expression, with progeny from low weight selected sires (LWS) having greater expression than progeny from high weight selected (HWS) sires (P = 0.0008). There was no difference between intestinal segments in progeny from HWS sires, however, greatest PepT1 gene expression was seen in the ileum of progeny from LWS sires (P < 0.0001). Overall, expression of EAAT3 was greatest in the ileum, intermediate in the jejunum and least in the ileum (P < 0.0001) and there was a segment by age interaction for EAAT3 expression (P < 0.0001). In all MCs except HH, EAAT3 gene expression increased from duodenum to ileum (P < 0.08). Within the ileum, the LL had greatest EAAT3 gene expression, LH and HL had intermediate gene expression, and HH had least expression (P < 0.08). Expression of SGLT1 gradually increased through D7 and decreased by D14 (P < 0.0001) and overall, was greatest in the distal small intestine (P < 0.0001). There was a MC by segment interaction, with SGLT1 gene expression being greatest in the distal small intestine in LL, LH, and HL, but greatest in the jejunum of HH (P < 0.04). Within the ileum, LL had greater SGLT1 gene expression than HH (P < 0.06). Overall, greatest GLUT5 expression was in the distal small intestine (P < 0.0001) and there was a MC by segment interaction, with expression being greatest in the distal small intestine in LL and HL (P < 0.02), greatest in the ileum of LH (P < 0.08), and greatest in the jejunum of HH (P < 0.09). Within the ileum there was a MC difference (P < 0.07). These results indicate that selection for high or low juvenile body weight may have influenced the gene expression pattern of these nutrient transporters in the small intestine, which may contribute to the overall differences in the growth and development of these lines of chickens. / Master of Science

The alpaca is a small South American camelid (SAC) that is an important production animal in Peru, especially among the highly impoverished communities of the high Andes, and raised for its fiber and meat. Alpacas are highly reliant on the microbes within their digestive tracts to digest the plant material they consume; volatile fatty acids (VFAs) are released as a byproduct of this microbial fermentation and used as a major source of energy by the alpaca. To explore optimal parameters for alpaca microbiome analysis, performed 16S rRNA gene surveys on alpaca C1 and fecal samples that had been extracted using one of three different DNA extraction methods (PowerFecal® DNA Isolation Kit (MO BIO); ZR Fecal DNA MiniPrep™ (Zymo); and a non-commercial extraction method called salting out) and amplified using one of two different polymerase enzyme mixes (AccuPrime™ Pfx SuperMix and 5 PRIME HotMasterMix). We found that choice of polymerase enzyme had a profound effect on the recovered microbiome, with the majority of 5 PRIME-amplified fecal samples failing to amplify. Extraction method had an effect on the recovered microbiome of fecal samples (but not C1 samples), with samples extracted using the MO BIO kit and the salting out method recovering different communities. The Zymo extraction kit returned microbial communities comparable to each of the other extraction methods. These results suggested that the AccuPrime enzyme and either the MO BIO or Zymo kits were optimal for alpaca gut microbiome analysis. We also performed two 16S rRNA gene surveys, the first from alpacas fed either a grass hay (GH) or alfalfa hay (AH) diet, and the second a C1 survey of alpacas fed two-week periods of mixed grass hay plus one of four supplements. We discovered body site and diet effects on the microbiota of alpacas fed either the GH or AH diet, with samples grouping by general body site (C1, small intestine, and distal intestine) and diet. However, we found no significant effect on the C1 microbiome of alpacas administered grain supplements. To study how energy extraction related to the microbiome, we correlated OTUs from GH/AH-fed alpaca with C1 VFA abundances. We discovered no significant correlations, and a 16S survey of low body condition (LBC) and good body condition (GBC) alpacas showed no difference in C1 microbial communities. We concluded that the microbiota of the alpaca digestive tract follow trends seen in microbiome studies of ruminants, but found no evidence of a relationship between body condition, energy extraction, and the C1 microbiome in alpacas.

Obesity is pandemic. Pharmacological treatment development depends on modeling the regulation of feeding, particularly by free fatty acids (FFA). Most models have been employed in the rat in vivo, and show FFA-stimulated intestinal satiety signals are dependent on the fat’s acyl chain-length, involve cholecystokinin (CCK) secretion, and are mediated by vagal afferents. I hypothesized that an in vitro mouse model could be employed, with sensitivity to measure afferent responses to nutrient stimuli. Male C57BL/6N mice were killed, the intestine harvested en bloc, and a jejunal section dissected with neurovascular mesenteric arcade emanating centrally. The tissue was placed in a Krebs-superfused chamber, the lumen cannulated with the outlet open to drain, and Krebs or other mediators were continuously perfused intraluminally. The dissected afferent nerve was placed in a suction electrode for extracellular recording. Afferent responses to distension and the perfusion of mediators (e.g. CCK or FFA) were tested. Preparations from normal mice (no surgery), or from mice following chronic subdiaphragmatic vagotomy or sham operation, were used to assess vagal afferent contributions. Luminally-perfused CCK (100 nM) increased afferent firing. This response was abolished with the CCK-1 receptor antagonist lorglumide (10 µM). The short-chain fatty acid (SCFA) sodium butyrate (30 mM) potentiated firing. The long-chain fatty acid (LCFA) sodium oleate (1-300 mM) activated concentration-dependent firing (EC50=25.35 mM) that was significantly greater at 30 mM than that evoked by butyrate. Lorglumide (30 µM) abolished the oleate (30 mM) response. The L-type Ca2+ channel (LTCC) inhibitor nicardipine (3 µM), intraluminally, potentiated the oleate response, while bath application abolished it. Vagotomy attenuated the oleate response. Vagotomy abolished the intraluminal CCK (100 nM) response, and attenuated the response to bath-superfused CCK. These findings support FFA chain-length-dependent mesenteric afferent activation and CCK involvement in oleate-induced firing, and suggest LTCC mediation of excitatory and inhibitory oleate response transduction pathways. The murine oleate response was shown to be mostly vagally-mediated, with some spinal contribution, and both vagal and spinal contributions to CCK responses were suggested. These data provide a basis for further investigation in vitro of cellular and molecular mechanisms of afferent satiety signals, and ultimately of obesity pathogenesis. / Thesis (Master, Physiology) -- Queen's University, 2010-06-29 15:56:08.387

The present study sought to determine whether a dietary and multi-element psychological treatment (DMPT) approach in combination with standard medical treatment would offer a more efficacious therapeutic package to irritable bowel syndrome (IBS) patients than would standard medical treatment (SMT) employed alone. The DMPT group (N = 19) received a stress management training package for a 2 week period consisting of relaxation training, imagery, and bowel sound biofeedback training via a stethoscope, in addition to instructions to increase their daily consumption of dietary fiber. They also were to continue the implementation of whatever standard medical treatment they were currently receiving, be it a bulking agent, or anti-anxiety, anti-cholinergic, or anti-depressant medications, etc. The SMT group (N = 19) simply received whatever conventional medical treatment they had been prescribed.

Background: The use of stapling equipment for intestinal surgery in cats is rarely reported, and appropriate staple sizes for cat intestine are unknown. Objective: To determine staple cartridge sizes for thoracoabdominal (TA) and endoscopic gastrointestinal anastomosis (EndoGIA) that will simultaneously prevent leakage of small intestinal contents while also allowing for sufficient vascular permeability past the staple lines for intestinal healing. Methods: Two sizes of EndoGIA cartridges (2.0/2.5/3.0mm and 3.0/3.5/4.0mm) and two sizes of TA cartridges (2.5mm and 3.5mm), applied in a transverse manner across fresh cadaveric cat jejunum, were evaluated via intestinal burst pressure testing for maximum intraluminal pressure prior to leaking, and via infusion of an intravascular dye at normal arterial pressures to determine percentage of vascular patency past the staple lines. Vascular patency was compared not only from pre-and post-staple segments of the same intestinal sample, but also EndoGIA vascular patency was evaluated against TA vascular patency. Results: Two cats met study criteria. All samples had intraluminal burst pressures over twice the chosen minimum (of 30mmHg). Vascular patency post- staple line ranged from 0-90.8%, with the most consistently high numbers noted with the TA 3.5mm cartridges. No EndoGIA cartridge had a post- staple line vascular patency higher than 31.1%, and no intravascular dye was noted in any post- staple line sample in the EndoGIA 2.0/2.5/3.0mm group. Conclusions: While statistical analysis of the dataset was unable to be performed due to low numbers of samples for comparison, both intestinal intraluminal burst pressure trends and intravascular dye patterns suggested both the TA 3.5mm cartridge and (to a lesser extent) the 3.0/3.5/4.0mm EndoGIA cartridge could provide the ideal combination of intraluminal seal without restriction of vascular access for healing. The intravascular dye infusion technique, developed during this research, shows promise as a future instrument to determine vascular patterns around intestinal implants in cadaveric cat specimens. / Master of Science / Despite the regularity of feline small intestinal surgery, few reports exist of stapled anastomoses in cats, in part due to stapler size limitations. However, the recently developed endoscopic gastrointestinal anastomosis (EndoGIA) stapler shows promise as a future surgical tool for cats because it fits into cat intestine. In dogs, 3.5mm staples are often chosen for intestinal surgery; however, dog intestine is considerably thicker than cat intestine. The study goal was to evaluate not only intestinal burst pressures (the pressure at which repaired intestine leaks), but also the ability of fluids to flow through blood vessels that cross the staple lines of four stapler cartridge types from two staple lineages (EndoGIA 2.0/2.5/3.0mm, EndoGIA 3.0/3.5/4.0mm, TA 2.5mm, and TA 3.5mm). The central hypothesis was twofold. First, smaller stapler cartridge sizes (the EndoGIA 2.0/2.5/3.0mm and TA 2.5mm) would have higher intraluminal burst pressures when compared to the larger sizes (the EndoGIA 3.0/3.5/4.0mm). Second, larger stapler cartridges (the EndoGIA 3.0/3.5/4.0mm and the TA 3.5mm) would allow for increased flow of fluids in blood vessels past the stapler lines compared to the smaller cartridges (the EndoGIA 2.0/2.5/3.0mm and the TA 2.5mm). Two cats were included in the study. Trends in the data suggested that all components of the hypothesis might be proven with further data. However, due to the low number of cats acquired during the study period, the hypotheses could not be verified with statistics. The dye infusion technique to evaluate flow of fluids in blood vessels, developed during this research, shows promise as a future instrument to determine vascular patterns around intestinal implants. Future research should focus on acquiring more cats to have the ability able to perform statistical analyses (and prove the hypothesis), before proceeding with additional related studies.

Studies investigating the effect of 5-HT receptors mediating a response in the neonatal intestine have been limited. There are evidences that the development of new neurones continues past postnatal term and this suggests that receptors expression may differ during maturation. Thus, 'in vitro' experiments were carried out to investigate the effects of ACh, atropine, 5-HT and its related drugs on intact intestinal segments taken from the ileum of adult and neonate rats. The application of ACh (3nM-1mM) and 5-HT (3nM-1mM) induced contractions in a concentration dependent manner in all tissues examined. The 5-HT induced contractions were only sensitive to antagonism by atropine (1μM) in segments taken from the neonates but not adults. The pre-treatment with methysergide (5-HT1/2/5-7 receptor antagonist), ritanserin (5-HT2 receptor antagonist), granisetron (5-HT3 receptor antagonist) and RS 23597 (5-HT4 receptor antagonist) at 1μM or a combination of ritanserin, granisetron, plus RS 23597 at 1μM significantly reduced or abolished contractile responses induced by 5-HT. SB 269970A (5-HT7 receptor antagonist) and WAY 100635 (5-HT1A receptor antagonist) at 1μM failed to influence contractile responses induced by 5-HT or the challenges to 5-HT receptor agonists, 5-CT (5-HT1A/7 receptor agonist) and 8-OH-DPAT (5-HT1A receptor agonist) at a concentration range of 10nM-0.1mM, indicating the unlikely involvement of 5-HT1A and 5-HT7 receptors in the mediation of contractile responses in the neonatal rat ileum. Results indicate differences in cholinergic receptor involvement during postnatal maturation and suggest the involvement of 5-HT2, 5-HT3 and 5-HT4 receptors in the mediation of contractile responses to 5-HT in the neonatal rat ileum. There is a growing need to decrease animal usage in pharmacological experiments. This may be achieved by the development of 'in vitro' cell culture models. Thus attempts were also made to develop a cell culture model of neonatal intestine to further investigate the action of pharmacologically active agents. The isolation of individual cell populations from segments taken from the intestine of rat neonates were achieved by ligation of both ends of the intestine prior to incubation in trypsin so that a gradual dissociation could be monitored. This was supported by histological procedures, determining the time required to extract large numbers of cells from different intestinal layers. Differential adhesion and selective cytotoxicity techniques were used for further purification of intestinal smooth muscle cells (ISMC), neuronal cells, and a coculture of ISMC and neuronal cells, and these were characterised through immunostaining with antibodies to α-smooth muscle actin, α-actinin and the 5-HT3 receptor. A protocol for cryopreservation of ISMC was designed in order to protect cells against genetic instability, enhance cell availability and reduce animal usage. Results showed that cells extracted from the intestine are viable for up to 4-months. ISMC functionality was analysed via the application of known pharmacologically active drugs on ISMC, which were plated onto glass and silicone elastomer substrate. The cultured ISMC responded to the application of drugs such as potassium chloride (KCl), carbachol, 5-HT and noradrenaline (NA). Large population of cocultures seeded onto silicone elastomers or cholesteric liquid crystal substrates (LC) were assessed for their ability to produce a collective response to KCl application. Attempts were made to detect any deformations of the substrate surface due to the exposure to KCl and NA. Cholesteric LC substrates seemed to be the most suitable material for investigating the cellular tensions. The availability of cell cultures allowed the development of an intestinal model of inflammation. This was achieved through the use of lipopolysaccharide (LPS)-induced inflammation and was confirmed by assessing the levels pro-inflammatory mediators interleukin (IL-8) and nitric oxide (NO), which were significantly elevated. Reduction of IL-8 ad NO was also examined using granisetron and L-NAME and Chaga mushroom extract. Granisetron and L-NAME reduced the NO production during short incubation times. However, an elevated level of NO was observed when longer treatment times were examined. The Chaga mushroom extract caused a significant reduction in NO production in the model of inflammation. This indicates that this model may be a valuable tool for the investigation of other pro-inflammatory mediators and may contribute for the investigation of more selective drugs in the management of intestinal inflammation in neonates.

published_or_final_version / abstract / Surgery / Doctoral / Doctor of Philosophy

<p>Malignant tumours of the small intestine are rare. Age-standardised incidence in Europe is between 0.5-1.5 per 100 000. As the small intestine represents more than 90 % of the gastrointestinal mucosal surface, it is surprising that it gives rise to less than 2 % of gastrointestinal malignancies. The dominating histological subtypes are carcinoids and adenocarcinomas. </p><p>We used three population-based registries in Sweden to study survival, second malignant tumours, causes of death, and Crohn’s disease as a risk factor for small intestinal adenocarcinoma and carcinoid.</p><p>We evaluated tumour site, sex, age, and year of diagnosis as prognostic factors. For adenocarcinomas there was no difference in survival between duodenal and jejunal/ileal tumours. Women with jejunal/ileal adenocarcinomas showed higher probabilities of survival than men, while no such relation was found for duodenal tumours. Old age correlated with poor survival for duodenal tumours, and prognosis has improved in later years. For carcinoids, duodenal tumours had a more favourable prognosis than jejunal/ileal tumours. There was no difference in survival between sexes. Old age correlated with poor survival, and survival has improved in recent years.</p><p>Female patients with adenocarcinoma had increased risk of acquiring cancer in the genital organs and breasts, and both sexes had increased risks of second tumours in the gastrointestinal tract and skin. Men with carcinoid tumours had increased risk of prostate cancer. Both sexes had increased risk of malignant melanoma and malignancies of endocrine organs.</p><p>Patients with adenocarcinoma had increased risk of dying from malignant diseases other than the primary small intestinal cancer and from gastrointestinal disease. The cohort with carcinoid had higher than expected risk of dying from malignant disease, gastrointestinal disease, and cardiovascular disease.</p><p>Patients with Crohn’s disease had increased risk of small intestinal adenocarcinoma and carcinoid, and the risk has increased for patients diagnosed in recent years.</p>