Intestinal model of Inflammation in primary cells

The gastrointestinal tract contains an enormous mucosal surface, which is continuously exposed to antigens hence making it susceptible to an inflammatory response. Such response targets potential pathogens by direct activation of the mucosal immune cells, however, in newborns the continuous inflammation attacks the intestine which leads to induction of necrotising enterocolitis. The present study aims at developing an “in-vitro” intestinal model of inflammation to assist in understanding the complex interplay of pro-inflammatory mediators during the immune response in neonates. Segments (1.5cm length) from the ileum were obtained from SD rat neonates (1-4 days old) and exposed to 0.25% trypsin/EDTA for 30min. Following trituration and subsequent centrifugation for 5min at 450xg, cells were suspended in DMEM-Hepes supplemented with 10% FCS, 2.5% Penicillin/Streptomycin, 2.5% L-Glutamine, and 0.2% Amphotericin B. Cell suspension were transferred to culture flaks and incubated at 37°C. Once confluent, the cell preparation media was replaced by FCS-free media, and treated with 0, 10, 50, and 100'g/ml of LPS. IL-8 and nitric oxide (NO) response were subsequently measured. In separate studies cell proliferation, cell viability, and cell adhesion were analysed. Additionally, the phenotypic properties of the intestinal muscle cells were also investigated via immunocytochemistry. Initial studies demonstrated that LPS treatments induced a significant increase in the release of IL-8 and NO compared to controls. The effect of LPS treatments on cell dynamics demonstrated small changes in cell viability and adhesion, whereas an increase in cell proliferation was observed. Immunocytochemistry studies indicated that LPS treatment caused a decrease in the expression of actin fibers with impaired distribution compared to controls. In the present model key aspects of intestinal inflammation were replicated “in-vitro” including the activation of pro-inflammatory mediators, the loss in enteric innervations and subsequent tissue hyperplasia. Thus, this model may be used as a tool to investigate the anti-inflammatory properties of candidate drugs targeting functional GI diseases