Epithelial restitution defect in neonatal jejunum is rescued by juvenile mucosal homogenate in a pig model of intestinal ischemic injury and repair

Abstract

21 22 2 23 ABSTRACT24 Intestinal ischemic injury results sloughing of the mucosal epithelium leading to host sepsis and 25 death unless the mucosal barrier is rapidly restored. Neonatal necrotizing enterocolitis (NEC) and 26 volvulus in infants is associated with intestinal ischemia, sepsis and high mortality rates. We have 27 characterized intestinal ischemia/ repair using a highly translatable porcine model in which 28 juvenile (6-8-week-old) pigs completely and efficiently restore barrier function by way of rapid 29 epithelial restitution and tight junction re-assembly. In contrast, separate studies showed that 30 younger neonatal (2-week-old) pigs exhibited less robust recovery of barrier function, which may 31 model an important cause of high mortality rates in human infants with ischemic intestinal disease.32 Therefore, we aimed to further refine our repair model and characterize defects in neonatal barrier 33 repair. Here we examine the defect in neonatal mucosal repair that we hypothesize is associated 34 with hypomaturity of the epithelial and subepithelial compartments. Following jejunal ischemia in 35 neonatal and juvenile pigs, injured mucosa was stripped from seromuscular layers and recovered 36 ex vivo while monitoring transepithelial electrical resistance (TEER) and 3 H-mannitol flux as 37 measures of barrier function. While ischemia-injured juvenile mucosa restored TEER above 38 control levels, reduced flux over the recovery period and showed 93±4.7% wound closure, 39 neonates exhibited no change in TEER, increased flux, and a 11±23.3% increase in epithelial 40 wound size. Scanning electron microscopy revealed enterocytes at the wound margins of 41 neonates failed to assume the restituting phenotype seen in restituting enterocytes of juveniles.42 To attempt rescue of injured neonatal mucosa, neonatal experiments were repeated with the 43 addition of exogenous prostaglandins during ex vivo recovery, ex vivo recovery with full thickness 44 intestine, in vivo recovery and direct application of injured mucosal homogenate from neonates 45 or juveniles. Neither exogenous prostaglandins, intact seromuscular intestinal layers, nor in vivo 46 recovery enhanced TEER or restitution in ischemia-injured neonatal mucosa. However, ex vivo 47 exogenous application of injured juvenile mucosal homogenate produced a significant increase 48 in TEER and enhanced histological restitution to 80±4.4% epithelial coverage in injured neonatal 3 49 mucosa. Thus, neonatal mucosal repair can be rescued through direct contact with the cellular 50 and non-cellular milieu of ischemia-injured mucosa from juvenile pigs. These findings support the 51 hypothesis that a defect in mucosal repair in neonates is due to immature repair mechanisms 52 within the mucosal compartment. Future studies to identify and rescue specific defects in neonatal 53 intestinal repair mechanisms will drive development of novel clinical interventions to reduce 54 mortality in infants affected by intestinal ischemic injury.55 56 INTRODUCTION 57 The intestina...

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

“…3 B, middle panels). When comparing the ischemic condition across age groups, "epithelial to mesenchymal transition," "apical junction" and "integrin-cell surface interactions" were negatively correlated with the neonates, enriched in juveniles, consistent with the defective epithelial barrier and defective epithelial restitution observed in this age group (Figure 3 B, right panels) (19). These results agree with the cellular phenotypes observed in neonates and juveniles, further demonstrating the lack of a coordinated transcriptional response to epithelial barrier injury in neonates as compared to juveniles.…”

“…As we have previously reported, the neonatal defect in restitution is rescued by direct application of ischemia-injured jejunal homogenate from juvenile-aged pigs, but the component of this homogenate which drives restitution has not yet been identified (19). Given the emerging and growing appreciation for the critical role of EGC in regulating and relaying signaling events in the intestinal mucosa in both homeostatic and pathologic conditions, we have considered their influence in our model.…”

“…Importantly, we have found that neonatal restitution can be rescued by direct application of ischemic-injured homogenized mucosa from juvenile pigs, suggesting that neonatal epithelial cells are intrinsically capable of efficient restitution in response to yet uncharacterized cues present in juvenile mucosa. (19) Therefore the identification of the components of the juvenile mucosal tissues which rescue neonatal restitution will inform future development of novel interventions for devastating intestinal diseases associated with age-dependent defects in barrier repair mechanisms. A complex and dense population of mesenchymal, circulatory, immune, and neural cells within the lamina propria are known to modulate epithelial cell functions in homeostasis and disease states by secreting paracrine factors to orchestrate complex, coordinated signaling in the mucosal microenvironment.…”

“…We recently found that in neonatal (2-week-old) pigs the epithelial barrier failed to restitute under the same conditions. (19) An age-dependent defect in epithelial restitution in neonates is likely to be a critical contributor to the high morbidity and mortality seen in infant patients clinically affected by intestinal ischemia and barrier injury. Therefore, this newly identified age-dependent intestinal repair defect in a highly comparative porcine model can provide critical insight for addressing the unacceptably high morbidity and mortality rates suffered by the youngest of critically ill patients.…”

“…Importantly, we have found that neonatal restitution can be rescued by direct application of ischemic-injured homogenized mucosa from juvenile pigs, suggesting that neonatal epithelial cells are intrinsically capable of efficient restitution in response to yet uncharacterized cues present in juvenile mucosa. (19) Therefore the identification of the components of the juvenile mucosal tissues which rescue neonatal restitution will inform future development of novel interventions for devastating intestinal diseases associated with age-dependent defects in barrier repair mechanisms.…”