Paediatric intestinal failure following bowel resection causes significant morbidity and mortality. There is a pressing need for improved treatment modalities. Following loss of bowel, the remaining intestine undergoes a period of adaptation, characterised by an increase in height of the intestinal villi. Better understanding the factors that govern the formation and growth of villi may lead to therapeutic interventions that amplify the intrinsic adaptation response. This thesis aims to explore the processes by which intestinal villi form during embryological development, the contribution of intestinal stem cells to this, and candidate signalling pathways that may yield insights into new therapeutic interventions for patients with intestinal failure. Abstract: Aim I will examine the morphogenesis and morphology of intestinal villi by investigating three themes: 1) Villus morphogenesis: When and where do villi form along the gut tube? Can this process be quantified, both in vivo and in vitro? Is this initiated by a dominolike signaling-cascade along the bowel, or location-specific intrinsic triggers? 2) Stem cells: What is the spatiotemporal appearance of the Lgr5-expressing intestinal stem cells during development? How does this relate to the process of villus morphogenesis? 3) Signalling pathways: Can a genetic mutation mouse model help elucidate pathways by which post bowel resection adaptation might occur? Can this be used to help identify potential intestinotrophic agents? Abstract: Materials and Methods Three mice models were used as the foundation for this work. Embryonic tissue was analysed from wild-type CD1 and Lgr5-eGFP-IRES-CreERT2 mice, and adult intestinal tissue examined from tamoxifen-activated Villin-Cre-ERT2 Pten-/- Brafv600E mice. Culture of wild-type embryonic mouse intestine with and without segments removed and / or reversed was performed to investigate the question of what triggers the proximal-to-distal wave of villus morphogenesis. Immunohistochemical interrogation using anti-GFP antibodies was used in the Lgr5- GFP mice to identify the location of Lgr5-expressing cells during the development of villi. Bright-field microscopy, time-lapse in-incubator microscopy, and histological sections assessed villus morphology. The Villin-Cre-ERT2 Pten-/- Brafv600E mouse mutant was explored regarding the intestinal epithelial morphometric changes that occur following tamoxifen-induction. Abstract: Results The proximal-to-distal wave of villus morphogenesis was observed both in vivo and in vitro. Villus morphogenesis commences at embryonic day 14.5 in vivo and after three days in culture from e11.5 in vitro. The villus structures formed in vitro are significantly attenuated compared to in vivo development. An attempt was made to overcome this by providing intestinal explants with a blood supply to aid growth. Evidence is presented that suggest the proximal-to-distal wave of villus morphogenesis is driven by location specific factors intrinsic to each part of the bowel, rather than a domino-like signalling cascade travelling along the intestine. Lgr5-expressing intestinal stem cells were present in early development. Prior to villus morphogenesis they were uniformly distributed along the luminal surface of the intestinal epithelia. During the intense proliferation associated with villus morphogenesis they progressively congregated to the inter-villus spaces. Once villi are fully formed they were absent from the villi but identified in the inter-villus spaces. The Pten/Braf mouse mutant demonstrates villus morphological changes similar to those found following post-bowel resection adaptation. This suggests that there may be a role for Pten/Braf in the epithelial proliferation following extensive bowel resection. Signalling factors in these pathways may be candidate intestinotrophic agents for the treatment of short bowel syndrome. Abstract: Conclusions Before any processes that manipulate intestinal epithelia can be safely translated into therapies to aid adaptation in patients with intestinal failure, it is important to have a full and detailed understanding of the basic science principles that underpin the behaviour of the epithelial cells, both during development and in adulthood. I have explored and quantified the process of villus morphogenesis in the embryonic mouse, investigated the timing of appearance of Lgr5 intestinal stem cells, and interrogated a genetic mouse model with morphometric changes similar to those seen following small bowel resection. I propose two candidate intestinotrophic agents that may hold regenerative potential to augment post small bowel resection adaptation. The next stage of investigation would be to use a mouse model of small bowel resection with manipulation of cell signalling factors to assess impact on post resection adaptation. The ultimate goal would be to investigate epithelial activity in human neonatal intestine and explore methods of modulating this to improve the outcomes from post bowel resection intestinal failure.
| Identifer | oai:union.ndltd.org:bl.uk/oai:ethos.bl.uk:738915 |
| Date | January 2017 |
| Creators | Partridge, Roland William |
| Contributors | Davies, Jamie ; Mason, John |
| Publisher | University of Edinburgh |
| Source Sets | Ethos UK |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
| Source | http://hdl.handle.net/1842/28884 |
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