(35) Early Life Antibiotic Exposure Impairs Postnatal Enteric Nervous System Maturation

Background: The enteric nervous system (ENS) regulates intestinal motility and is organized into two plexuses: the myenteric and the submucosal. Enteric precursor cells populate the myenteric layer first and then are induced to radially migrate luminally by the chemoattractant netrin-1 to form the submucosal plexus. ENS development continues through the first weeks of postnatal life, especially in the submucosal plexus.

Objectives: As early life antibiotic exposure and dysmotility are both associated with increasing the risk of gastrointestinal complications of prematurity, we hypothesize that antibiotic-induced dysbiosis could disrupt neonatal ENS development

Design/Methods: We exposed postnatal day (P) 1-8 mice to ampicillin and neomycin via maternal drinking water. Motility was assessed with a FITC-dextran dye transit assay. Mid-colon tissue was isolated and the muscularis layer was dissected from the submucosal layer. Nestin-creERT2 expression was induced by intraperitoneal tamoxifen treatment on P5 and 7. Immunofluorescence staining and ELISA were performed on muscularis and submucosal layers individually.

Results: Antibiotic-exposed specific pathogen free (SPF) pups had increased neuronal density in the colonic myenteric plexus on P8 and a concomitant slowing of intestinal motility compared to controls. To understand how dysbiosis impacts gene expression of developing enteric neurons, we performed neuron specific translating ribosome affinity purification and bulk sequencing. Colonic enteric neurons from antibiotic-exposed pups had reduced expression of genes associated with mature neurons. Consistent with our sequencing data, neurons from antibiotic exposed mice had decreased density of Tuj1 (a neuron specific tubulin) and fewer synaptophysin puncta (a synaptic marker) as measured by by immunofluorescence. To determine if radial migration was impacted by antibiotic exposure, we used fate mapping to quantify the ENS progenitor cells in the submucosal plexus. There was a significant decrease in nestin positive ENS progenitor cells present in the submucosal plexus of antibiotic exposed mice compared to controls. Further, levels of the chemoattractant netrin-1 in the submucosal/epithelial colonic tissue was significantly lower netrin-1 levels in antibiotic exposed mice compared to controls.

Conclusion: These data demonstrate that neonatal dysbiosis impairs colonic radial migration leading to impaired neuronal maturation in the ENS. Further studies are ongoing to determine how this ENS disruption contributes to the increased risk of gastrointestinal complications of prematurity induced by antibiotic exposure.