Characterization of proliferative cells in developing and adult rats after early weaning.

Author(s)

Isadora Campos Rattes

• Document type: Doctoral Thesis
• Press: São Paulo.
• Institution: Universidade de São Paulo (USP). Instituto de Ciências Biomédicas (ICB/SDI)
• Defense date: 2023-09-05

Examining board members

Patricia Gama; João Paulo Fabi; Luciana Harumi Osaki; Marinilce Fagundes dos Santos

• Advisor: Patricia Gama

Abstract

Proliferative cells are found throughout the gland during postnatal development of the rat and, after weaning, are concentrated in the isthmus and neck region. In adult animals, an additional niche is also found at the base of the gland, but the characterization of markers for stem/proliferative cells is still discussed in the literature and little is known about the activity of these cells during postnatal development, and the organization of the environment, in terms of growth factors and signaling, and how the feeding pattern can affect these elements and influence the maturation of the gland is unknown. The breastfeeding period is critical for the maturation of the stomach in terms of nutrient supply and growth factors, so abrupt cessation of breastfeeding may be detrimental to the development of the organ. Thus, our hypothesis was that abrupt dietary change with early weaning entails perturbations in the proliferative niche that may extend into adulthood. Our aim was to evaluate how early weaning regulates proliferative activity in terms of distribution and dynamics of cell kinetics, the expression of genes important in the regulation of proliferative niches, and to evaluate the presence of Troy (reserve stem cell marker) in the the gastric epithelium of rats. On day 15, Wistar rat pups were divided into two groups: suckling (S) and early weaning (EW). The S animals remained with their mothers and were breastfed until day 21. The EW rats were separated from their mothers and kept in another cage, where the diet became a paste of hydrated feed. Gastric mucosa samples were collected at 15, 18, 30, 60 and 120 days for analysis of morphology, gene expression by RT-qPCR and protein levels by immunohistochemistry and Western Blot. The results indicated a distinct distribution of Ki-67+ proliferative cells between 18-day S and EW, which showed a similar profile to adults. In addition, EW led to increased proliferation at the base of the gland in the 60-day-old animals, but no differences were observed at 120 days. Gland growth dynamics vary with advancing age after EW, and cell kinetics were studied by BrdU and EdU incorporation. The distribution of BrdU+, EdU+ and BrdU/EdU+ cells varied between S and EW and across ages, indicating specific effects of EW on cell cycle entry. Regarding gene expression, EW decreased Wnt3, Notch1, Notch2, and increased Bmp2 at 18 days. At 60 days, EW increased Axin2, Notch1 and Notch2, with no differences at 120 days. In silico analysis indicated the presence of Notch, BMP, Wnt and SHH pathway components differentially expressed in zymogenic and parietal cells. As for Notch1+ cells, EW decreases the number of labelled cells at 60 days and Notch2+ cells increase the number of cells in the isthmus and decreases at the base at 60 days. Regarding the Troy marker, we found that from day 3 to 15 the number of Troy+ cells does not differ, and from day 18 the number of Troy+ cells increase in the gastric mucosa. In protein levels, EW increases Troy at 18 days, but at 60 days there is a reversal with a decrease in these levels. We conclude that EW causes immediate and delayed effects on proliferation and stem cell markers in the gastric mucosa, modulating important genes in proliferative niche regulation pathways. (AU)

• FAPESP's process: 18/05064-0 - Characterization of the stem cell niche in the gastric mucosa of developing and adult rats and the role of early weaning on this population
• Grantee: Isadora Campos Rattes
• Support Opportunities: Scholarships in Brazil - Doctorate (Direct)