Conclusions
AO enteroids present an innovative model for NEC with increased inflammation and gut barrier restructuring. This model allows for a biologically relevant investigation of the interaction between the pathogen and the intestinal epithelial barrier in NEC.
Methods
Following the institutional review board approval (#11610-11611), neonatal intestinal tissue was collected from surgical specimens. Stem cells were collected; enteroids were generated and grown to maturity in BO conformation then everted to AO. Enteroids were untreated or treated for 24 h with 100 μg/mL lipopolysaccharide and hypoxia. Protein and gene expression were analyzed for inflammatory markers, tight junction (TJ) proteins and permeability characteristic of NEC.
Results
Apical TJ protein zonula occludens-1 and basolateral protein β-catenin immunofluorescence confirmed AO configuration. Treated AO enteroids had significantly increased messenger RNA (P = 0.001) and protein levels (P < 0.0001) of tumor necrosis factor-α compared to controls. Corrected total cell fluorescence of toll-like receptor 4 was significantly increased in treated AO enteroids compared to control (P = 0.002). Occludin was found to have significantly decreased messenger RNA in treated AO enteroids (P = 0.003). Expression of other TJ proteins claudins-1, -4 and zonula occludens-1 was significantly decreased in treated AO enteroids (P < 0.05). Conclusions: AO enteroids present an innovative model for NEC with increased inflammation and gut barrier restructuring. This model allows for a biologically relevant investigation of the interaction between the pathogen and the intestinal epithelial barrier in NEC.