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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 (EGCs) 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 glial activity were examined by Gene Set Enrichment Analysis, three-dimensional (3-D) volume imaging, and Western blot and its function in regulating epithelial restitution was assessed ex vivo in Ussing chamber using the glia-specific inhibitor fluoroacetate (FA), and in vitro by coculture 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. Furthermore, we report important evidence that the subepithelial EGC network changes significantly over the early postnatal period and demonstrate that the proximity of a specific functional population of EGC to wounded intestinal epithelium contributes to intestinal barrier restitution following ischemic injury. NEW & NOTEWORTHY This 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 for enteric glial cell (EGC) 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.

Insulin-like growth factor 1 (IGF1) has potent trophic effects on normal or injured intestinal epithelium, but specific effects on intestinal stem cells (ISCs) are undefined. We used Sox9-enhanced green fluorescent protein (EGFP) reporter mice that permit analyses of both actively cycling ISCs (Sox9-EGFP(Low)) and reserve/facultative ISCs (Sox9-EGFP(High)) to study IGF1 action on ISCs in normal intestine or during crypt regeneration after high-dose radiation-induced injury. We hypothesized that IGF1 differentially regulates proliferation and gene expression in actively cycling and reserve/facultative ISCs. IGF1 was delivered for 5 days using subcutaneously implanted mini-pumps in uninjured mice or after 14 Gy abdominal radiation. ISC numbers, proliferation, and transcriptome were assessed. IGF1 increased epithelial growth in nonirradiated mice and enhanced crypt regeneration after radiation. In uninjured and regenerating intestines, IGF1 increased total numbers of Sox9-EGFP(Low) ISCs and percentage of these cells in M-phase. IGF1 increased percentages of Sox9-EGFP(High) ISCs in S-phase but did not expand this population. Microarray revealed that IGF1 activated distinct gene expression signatures in the 2 Sox9-EGFP ISC populations. In vitro IGF1 enhanced enteroid formation by Sox9-EGFP(High) facultative ISCs but not Sox9-EGFP(Low) actively cycling ISCs. Our data provide new evidence that IGF1 activates 2 ISC populations via distinct regulatory pathways to promote growth of normal intestinal epithelium and crypt regeneration after irradiation., (© FASEB.)

Background: In vivo studies of high dose radiation-induced crypt and intestinal stem cell (ISC) loss and subsequent regeneration are typically restricted to 5-8 days after radiation due to high mortality and immune failure. This study aimed to develop murine radiation models of complete crypt loss that permit longer-term studies of ISC and crypt regeneration, repair and normalization of the intestinal epithelium., Methods: In C57Bl/6J mice, a predetermined small intestinal segment was exteriorized and exposed to 14 Gy-radiation, while a lead shield protected the rest of the body from radiation. Sham controls had segment exteriorization but no radiation. Results were compared to C57Bl/6J mice given 14 Gy-abdominal radiation. Effects of elemental liquid diet feeding from the day prior to radiation until day 7 post-radiation were assessed in both models. Body weight and a custom-developed health score was assessed every day until day 21 post-radiation. Intestine was assessed histologically., Results: At day 3 after segment radiation, complete loss of crypts occurred in the targeted segment, while adjacent and remaining intestine in segment-radiated mice, and entire intestine of sham controls, showed no detectable epithelial damage. Liquid diet feeding was required for survival of mice after segment radiation. Liquid diet significantly improved survival, body weight recovery and normalization of intestinal epithelium after abdominal radiation. Mice given segment radiation combined with liquid diet feeding showed minimal body weight loss, increased food intake and enhanced health score., Conclusions: The segment radiation method provides a useful model to study ISC/crypt loss and long-term crypt regeneration and epithelial repair, and may be valuable for future application to ISC transplantation or to genetic mutants that would not otherwise survive radiation doses that lead to complete crypt loss. Liquid diet is a simple intervention that improves survival and facilitates long-term studies of intestine in mice after high dose abdominal or segment radiation.

Wound healing of the gastrointestinal mucosa is essential for the maintenance of gut homeostasis and integrity. Enteric glial cells play a major role in regulating intestinal barrier function, but their role in mucosal barrier repair remains unknown. The impact of conditional ablation of enteric glia on dextran sodium sulfate (DSS)-induced mucosal damage and on healing of diclofenac-induced mucosal ulcerations was evaluated in vivo in GFAP-HSVtk transgenic mice. A mechanically induced model of intestinal wound healing was developed to study glial-induced epithelial restitution. Glial-epithelial signaling mechanisms were analyzed by using pharmacological inhibitors, neutralizing antibodies, and genetically engineered intestinal epithelial cells. Enteric glial cells were shown to be abundant in the gut mucosa, where they associate closely with intestinal epithelial cells as a distinct cell population from myofibroblasts. Conditional ablation of enteric glia worsened mucosal damage after DSS treatment and significantly delayed mucosal wound healing following diclofenac-induced small intestinal enteropathy in transgenic mice. Enteric glial cells enhanced epithelial restitution and cell spreading in vitro. These enhanced repair processes were reproduced by use of glial-conditioned media, and soluble proEGF was identified as a secreted glial mediator leading to consecutive activation of epidermal growth factor receptor and focal adhesion kinase signaling pathways in intestinal epithelial cells. Our study shows that enteric glia represent a functionally important cellular component of the intestinal epithelial barrier microenvironment and that the disruption of this cellular network attenuates the mucosal healing process.

Although recent studies have shown that enteric neurons control intestinal barrier function, the role of enteric glial cells (EGCs) in this control remains unknown. Therefore, our goal was to characterize the role of EGCs in the control of intestinal epithelial cell proliferation using an in vivo transgenic and an in vitro coculture model. Assessment of intestinal epithelial cell proliferation after ablation of EGCs in transgenic mice demonstrated a significant increase in crypt cell hyperplasia. Furthermore, mucosal glial network (assessed by immunohistochemical detection of S-100beta) is altered in colon adenocarcinoma compared with control tissue. In an in vitro coculture model of subconfluent Caco-2 cells seeded onto Transwell filters with EGCs, Caco-2 cell density and [3H]thymidine incorporation were significantly lower than in control (Caco-2 cultured alone). Flow cytometry analysis showed that EGCs had no effect on Caco-2 cell viability. EGCs induced a significant increase in Caco-2 cell surface area without any sign of cellular hypertrophy. These effects by EGCs were also seen in various transformed or nontransformed intestinal epithelial cell lines. Furthermore, TGF-beta1 mRNA was expressed, and TGF-beta1 was secreted by EGCs. Exogenously added TGF-beta1 reproduced partly the EGC-mediated effects on cell density and surface area. In addition, EGC effects on Caco-2 cell density were significantly reduced by a neutralizing TGF-beta antibody. In conclusion, EGCs have profound antiproliferative effects on intestinal epithelial cells. Functional alterations in EGCs may therefore modify intestinal barrier functions and be involved in pathologies such as cancer or inflammatory bowel diseases.