Intestinal cell kinetics : Modulation caused by age, gender and microbial status in rats and mice. An experimental study in germfree, conventional, Lactobacillus rhamnosus GG and Clostridium difficile monoassociated animals

Detta är en avhandling från Stockholm : Karolinska Institutet, Department of Cell and Molecular Biology

Sammanfattning: Mammals living in symbiosis with microbes create conventional organisms. The interactions or cross-talks that continuously occur demonstrate how microbes may influence on series of parameters, including intestinal cell kinetics and mucosal morphology. The aim of this study was to investigate modulation of intestinal cell kinetics and morphological parameters caused by age, gender, and microbial status in rats and mice. These studies were performed in germfree (GF), conventional (Conv), and ex-GF animals, mono-associated with either Lactobacillus rhamnosus GG (LGG) or Clostridium difficile. The first two studies were performed in GF and Conv rats and mice, aiming to evaluate the influence of gender, age and microbial status on epithelial cell kinetic and morphological parameters in different compartments of the intestinal tract. These investigations were performed in order to establish base-lines for future cell kinetic and morphological investigations. The mitotic index, growth fraction, number of cells in the crypts and villi, depth of the crypts, height of the villi and crypt/villus ratio were determined. In main, the results were as followed; Rats: Gender: The effects were mainly expressed in the upper part of the small intestine (higher mitotic activity, more cells in the crypts and villi, and deeper crypts in males than in females). Age: The effects were mainly upon morphological parameters in the distal part of the small intestine (deeper crypts, longer villi and more cells in the villi in the older rats). Microbial status: The effects were somewhat scattered throughout the intestine. The most pronounced differences were longer villi and more cells in the villi in GF rats. Mice: In main, influences of gender and age were as found in rats. However, the influence of microbial status was more pronounced in mice, especially in the large intestine. Taken together, the results demonstrated that gender, age and microbial status influenced upon cell kinetics as well as intestinal mucosa morphology in rats and mice in a compartmentalised manner. The following two studies composed association of young GF male rats with either a probiotic strain or a potentially pathogenic strain, aiming to study the influence of these microbes on cell kinetic parameters. LGG is used for production of fermented food products i.e., probiotics, aiming to promote health. Cl difficile is suggested to be one major cause of antibiotic-associated diarrhea. The results from LGG inoculated rats showed that a short-term mono-association induced a compartmentalised increase in cell production and number of villus cells. Intestinal cell production is looked upon as a major regulatory and protective defence mechanism. It was assumed that this stimulatory effect could be of importance for explaining the protective and curative effects of LGG in rota-virus infections. The results from rats mono-associated with a toxin producing strain of Cl difficile showed a marked influence on cell kinetics. After 3 days mono-association, a significant increase in cell production was observed in all compartments, followed by a dramatic reduction on day 7 and a shift to Conv values on day 21. Histological investigations revealed weak signs of damages. The rats looked healthy throughout the study period and no signs of diarrhea were observed. Increasing amounts of toxin were found throughout the intestinal tract during the study. In another series of experiments, GF rats were mono-associated with a non-toxin producing strain of Cl difficile. A slight increase in intestinal cell production was seen after 3 days, but after 7 days no dramatic reduction was observed. Our putative theory is that toxin producing Cl difficile, by temporally switching off the intestinal cell production, creates suitable attack points for unknown pathogens in the intestinal flora to cause symptoms and signs related to the so-called antibiotic-associated diarrhea. Thus, intestinal microbes - physiological as well as pathophysiological strains - act on intestinal cell kinetics and morphology in a compartmentalised manner. Increased knowledge on such prokaryotic/eucariotic cross-talks creates possibilities for new prophylactic and therapeutic approaches. Gnotobiotic animals are irreplaceable models in these studies.

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