Touvron, M. | Search bioRxiv

bioRxiv2022

Enteric Glial Cell Network Function is Required for Epithelial Barrier Restitution following Intestinal Ischemic Injury in the Early Postnatal Period

Ischemic damage to the intestinal epithelial barrier, such as in necrotizing enterocolitis or small intestinal volvulus, is associated with higher mortality rates in younger patients. We have recently reported a powerful pig model to investigate these age-dependent outcomes in which mucosal barrier restitution is strikingly absent in neonates but can be rescued by direct application of homogenized mucosa from older, juvenile pigs by a yet-undefined mechanism. Within the mucosa, a postnatally developing network of enteric glial cells (EGC) is gaining recognition as a key regulator of the mucosal barrier. Therefore, we hypothesized that the developing EGC network may play an important role in coordinating intestinal barrier repair in neonates. Neonatal and juvenile jejunal mucosa recovering from surgically induced intestinal ischemia was visualized by scanning electron microscopy and the transcriptomic phenotypes were assessed by bulk RNA sequencing. EGC network density and gliosis were examined by gene set enrichment analysis, three-dimensional volume imaging and western blot and its function in regulating epithelial restitution assessed ex vivo in Ussing chamber using the glia-specific inhibitor fluoroacetate, and in vivo by co-culture assay. Here we refine and elaborate our translational model, confirming a neonatal phenotype characterized by a complete lack of coordinated reparative signaling in the mucosal microenvironment. Further, we report important evidence that the subepithelial EGC network changes significantly over the early postnatal period and demonstrate that EGC function in close proximity to wounded intestinal epithelium is critical to intestinal barrier restitution following ischemic injury. NEW & NOTEWORTHYThis study refines a powerful translational pig model, defining an age-dependent relationship between enteric glia and the intestinal epithelium during intestinal ischemic injury and confirming an important role of the enteric glial cell activity in driving mucosal barrier restitution. This study suggests that targeting the enteric glial network could lead to novel interventions to improve recovery from intestinal injury in neonatal patients.

physiology↗

bioRxiv2022

Enteric glial cells of the two plexi of the enteric nervous system exhibit phenotypic and functional inter-and intra-heterogeneity

Enteric glial cells (EGC) are a prominent cell type of all layers of the gut wall, virtually controlling all gastrointestinal functions. While the development of transgenic mice has led to major advances in understanding EGC biology, in vitro models are still fairly limited and do not allow for the robust and reproducible establishment of primary cultures discriminating EGC from the inner versus outer layers of the gut wall. Here we report a novel method to separately grow EGC from the inner and outer layers of the intestinal wall from the same mouse with a high degree of purity and cell heterogeneity. Our results indicate that EGC from the inner layers of the gut wall exhibit higher calcium response to ATP when compared to EGC from the outer layers. We also show that inner EGC cultures express lower levels of the transcription factor Sox 10 as compared to outer EGC cultures, which mirrors in situ differential expression of Sox10 in submucosal (inner) versus myenteric (outer) plexus assessed using wholemounts. Confocal microscopy analyses of wholemounts further demonstrate that a majority of calretinin-expressing ganglionic cells of the submucosal plexus express the EGC marker S-100{beta}, while this population is marginally represented in ganglia of the myenteric plexus. Altogether this study describes a novel method of EGC primary cultures permitting for the first time to compare inner versus outer EGC and provides in vitro and ex vivo evidence that inner EGC and outer EGC are phenotypically and functionally distinct.

neuroscience↗