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This paper supports that rISCs are resistant to ischemic injury and likely an important source of cellular renewal following near-complete epithelial loss. Furthermore, we have evidence that HOPX controls ISC activity state and may be a critical signaling pathway during ISC-mediated repair. Finally, we use multiple novel methods to evaluate ISCs in a translationally relevant large animal model of severe intestinal injury and provide evidence for the potential role of rISCs as therapeutic targets.

All species, including horses, suffer from alterations that increase intestinal permeability. These alterations, also known as "leaky gut," may lead to severe disease as the normal intestinal barrier becomes compromised and can no longer protect against harmful luminal contents including microbial toxins and pathogens. Leaky gut results from a variety of conditions including physical stressors, decreased blood flow to the intestine, inflammatory disease, and pathogenic infections, among others. Several testing methods exist to diagnose these alterations in both a clinical and research setting. To date, most research has focused on regulation of the host immune response due to the wide variety of factors that can potentially influence the intestinal barrier. This article serves to review the normal intestinal barrier, measurement of barrier permeability, pathogenesis and main causes of altered permeability, and highlight potential alternative therapies of leaky gut in horses while relating what has been studied in other species. Conditions resulting in barrier dysfunction and leaky gut can be a major cause of decreased performance and also death in horses. A better understanding of the intestinal barrier in disease and ways to optimize the function of this barrier is vital to the long-term health and maintenance of these animals.

Intestinal ischemia is an abdominal emergency with a mortality rate >50%, leading to epithelial barrier loss and subsequent sepsis. Epithelial renewal and repair after injury depend on intestinal epithelial stem cells (ISC) that reside within the crypts of Lieberkühn. Two ISC populations critical to epithelial repair have been described: 1) active ISC (aISC; highly proliferative; leucine-rich-repeat-containing G protein-coupled receptor 5 positive, sex determining region Y-box 9 positive) and 2) reserve ISC [rISC; less proliferative; homeodomain only protein X (Hopx)+]. Yorkshire crossbred pigs (8–10 wk old) were subjected to 1–4 h of ischemia and 1 h of reperfusion or recovery by reversible mesenteric vascular occlusion. This study was designed to evaluate whether ISC-expressing biomarkers of aISCs or rISCs show differential resistance to ischemic injury and different contributions to the subsequent repair and regenerative responses. Our data demonstrate that, following 3–4 h ischemic injury, aISC undergo apoptosis, whereas rISC are preserved. Furthermore, these rISC are retained ex vivo in spheroids in which cell populations are enriched in the rISC biomarker Hopx. These cells appear to go on to provide a proliferative pool of cells during the recovery period. Taken together, these data indicate that Hopx+ cells are resistant to injury and are the likely source of epithelial renewal following prolonged ischemic injury. It is therefore possible that targeting reserve stem cells will lead to new therapies for patients with severe intestinal injury. NEW & NOTEWORTHY The population of reserve less-proliferative intestinal epithelial stem cells appears resistant to injury despite severe epithelial cell loss, including that of the active stem cell population, which results from prolonged mesenteric ischemia. These cells can change to an activated state and are likely indispensable to regenerative processes. Reserve stem cell targeted therapies may improve treatment and outcome of patients with ischemic disease.

Intestinal ischemia remains a major cause of morbidity and mortality in human and veterinary patients. Many disease processes result in intestinal ischemia, when the blood supply and therefore oxygen is decreased to the intestine. This leads to intestinal barrier loss and damage to the underlying tissue. Intestinal stem cells reside at the base of the crypts of Lieberkuhn and are responsible for intestinal renewal during homeostasis and following injury. Ex vivo cell culture techniques have allowed for the successful study of epithelial stem cell interactions by establishing culture conditions that support the growth of three-dimensional epithelial organ-like systems (termed "enteroids" and "colonoids" from the small and large intestine, respectively). These enteroids are composed of crypt and villus-like domains and mature to contain all of the cell types found within the epithelium. Historically, murine models have been utilized to study intestinal injury. However, a porcine model offers several advantages including similarity of size as well as gastrointestinal anatomy and physiology to that of humans. By utilizing a porcine model, we establish a protocol in which segmental loops of intestinal ischemia can be created within a single animal, enabling the study of differing time points of ischemic injury and repair in vivo. Additionally, we describe a method to isolate and culture the intestinal stem cells from the ischemic loops of intestine, allowing for the continued study of epithelial repair, modulated by stem cells, ex vivo.