Esophageal/pyloric ligation enhances development of the murine fetal stomach in organ culture

Abstract

Purpose: The authors hypothesized that increased intraluminal pressure in the fetal stomach would enhance development in a murine organ culture model. Methods: Gestation day 14 (Gd14) fetal stomachs from time-dated pregnant CD-1 mice (term, 20 days) were maintained in organ culture for 7 days. Some stomachs were ligated at the gastroesophageal (GE) and pyloroduodenal (PD) junctions. Others were left unligated. Gd14, Gd16, and Gd18 stomachs were taken as well to compare organogenesis in vivo. Tissues were processed for histological, morphometric, and immunohistochemical analysis, as well as total protein and DNA determination. Results: The ligated stomachs were visibly distended compared with unligated stomachs in organ culture after 7 days. The length and width of the 7-day in vitro ligated stomachs were significantly increased compared with unligated (2.97 ± 0.04 mm v 2.48 ± 0.05 mm and 2.14 ± 0.04 mm v 1.57 ± 0.08 mm, respectively, P < .05). Mucosal epithelial cells showed nuclear polarization, and there was a distinct outer muscle layer in the ligated stomachs, but not in the unligated stomachs, which demonstrated pseudostratified epithelial cells in the mucosa. The ligated stomachs had increased in mucosal thickness compared with unligated (31.4 ± 1.3 μm vs 24.9 ± 0.9 μm, p < 0.05). The ligated stomachs also had significantly increased protein and DNA content when compared with unligated stomachs (65.8 ± 3.1 μg and 23.3 ± 1.2 μg v 55.0 ± 2.7 μg and 19.0 ± 1.2 μg, respectively, P < .05). However, there were no significant differences noted between the protein to DNA ratios. Immunohistochemical staining for proliferating cell nuclear antigen (PCNA), a marker for cell proliferation, demonstrated increased proliferative activity of the mucosal epithelial cells in the ligated stomachs. Conclusions: Esophageal and pyloric ligation enhanced the development of the fetal stomach in vitro in comparison with unligated stomachs cultured under similar conditions. Developmental characteristics of the ligated stomachs paralleled that of Gd16 stomachs in vivo.