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Background & AimsHypoxia in the intestinal epithelium can be caused by acute ischemic events or conditions like Inflammatory Bowel Disease (IBD) where immune cell infiltration produces ‘inflammatory hypoxia’, a chronic condition that starves the mucosa of oxygen. Epithelial regeneration after ischemia and IBD suggests intestinal stem cells (ISCs) are highly tolerant to acute and chronic hypoxia; however, the impact of acute and chronic hypoxia on human ISC (hISC) properties have not been reported. Here we present a new microphysiological system (MPS) to investigate how hypoxia affects hISCs isolated from healthy human tissues. We then test the hypothesis that some inflammation-associated interleukins protect hISCs during prolonged hypoxia.MethodshISCs were exposed to ResultsThe novel MPS enables precise, real-time control and monitoring of oxygen levels at the cell surface. Under hypoxia, hISCs remain viable until 72hrs and exhibit peak HIF1α at 24hrs. hISCs lose stem cell activity at 24hrs that recovers at 48hrs of hypoxia. Hypoxia increases the proportion of hISCs in G1 and regulates hISC capacity to respond to multiple inflammatory signals. Hypoxia induces hISCs to upregulate many interleukin receptors and hISCs demonstrate hypoxia-dependent cell cycle regulation and increased organoid forming efficiency when treated with specific interleukinsConclusionsHypoxia primes hISCs to respond differently to interleukins than hISCs in normoxia through a transcriptional response. hISCs slow cell cycle progression and increase hISC activity when treated with hypoxia and specific interleukins. These findings have important implications for epithelial regeneration in the gut during inflammatory events.

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 assessedex vivoin Ussing chamber using the glia-specific inhibitor fluoroacetate, andin vivoby 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.

The intestinal epithelial barrier is susceptible to injury from insults, such as ischemia or infectious disease. The epithelium's ability to repair wounded regions is critical to maintaining barrier integrity. Mechanisms of intestinal epithelial repair can be studied with models that recapitulate the in vivo environment. This review focuses on in vitro injury models and intestinal cell lines utilized in such systems. The formation of artificial wounds in a controlled environment allows for the exploration of reparative physiology in cell lines modeling diverse aspects of intestinal physiology. Specifically, the use of intestinal cell lines, IPEC-J2, Caco-2, T-84, HT-29, and IEC-6, to model intestinal epithelium is discussed. Understanding the unique systems available for creating intestinal injury and the differences in monolayers used for in vitro work is essential for designing studies that properly capture relevant physiology for the study of intestinal wound repair.

OBJECTIVE To determine the degree of histomorphometric damage in dorsal colon and pelvic flexure biopsy specimens (DCBSs and PFBSs, respectively) obtained from horses with large colon volvulus (LCV) and assess the accuracy of predicting short-term outcome for those horses on the basis of DCBS or PFBS characteristics. ANIMALS 18 horses with ≥ 360° LCV that underwent large colon resection. PROCEDURES During surgery, biopsy specimens from the dorsal colon resection site and the pelvic flexure (when available) were collected from each horse. Interstitial-to-crypt (I:C) ratio (ratio of the lamina propria space occupied by the interstitium to that occupied by crypts), hemorrhage within the lamina propria (mucosal hemorrhage score [MHS] from 0 to 4), and percentage losses of glandular and luminal epithelium were determined in paired biopsy specimens and compared to determine optimal cutoff values for calculating the accuracy of DCBS and PFBS characteristics to predict short-term outcome (survival or nonsurvival after recovery from surgery). RESULTS Paired biopsy specimens were obtained from 17 of the 18 horses. The I:C ratio and percentage glandular epithelial loss differed between DCBSs and PFBSs. For DCBSs, an I:C ratio ≥ 0.9 and MHS ≥ 3 each predicted patient nonsurvival with 77.8% accuracy. For PFBSs, an I:C ratio ≥ I and MHS ≥ 3 predicted patient nonsurvival with 70.6% and 82.4% accuracy, respectively. CONCLUSIONS AND CLINICAL RELEVANCE Although different, histomorphometric measurements for either DCBSs or PFBSs could be used to accurately predict short-term outcome for horses with LCV that underwent large colon resection, and arguably PFBSs are easier to collect.

Orthopaedic disorders are commonly encountered in equine veterinary medicine, and non-steroidal anti-inflammatory drugs (NSAIDs) play an important role in the management of many equine orthopaedic disorders. There are multiple NSAIDs available for use in horses, including both non-selective and selective NSAIDS, and the body of literature evaluating the efficacy of these medications, their effects on normal and inflamed musculoskeletal tissues, and their side effects is broad. This review aims to summarise the current literature on the use of NSAIDs for equine orthopaedic disorders and examines new and future avenues for the management of inflammation in equine orthopaedics.

Intestinal barrier function is critical to prevent translocation of microorganisms and their toxins from the intestinal lumen to the circulation. This is particularly noteworthy in horses, in which colic may be associated with ischemia and breakdown of the intestinal barrier, and in pigs, in which infectious diseases such as PEDv cause epithelial soughing and loss of intestinal barrier function. The term ‘function’ refers to the ability of a single layer of columnar epithelium that lines the intestinal tract to form a physical and dynamic barrier. This layer of epithelium is linked by a series of interepithelial junctions, most importantly tight junctions, that has to prevent passage of noxious luminal contents, while simultaneously serving to transport electrolytes, water, and nutrients necessary for physiological function of the host. The tight junctions are dynamically regulated by the cytoskeleton via nutrient signaling, and perturbed by reduced oxygen tension and pathogenic organisms. Barrier function also includes the ability to rapidly repair. In the small intestine, villus contraction is the initial phase of repair, followed by crawling of epithelium adjacent to the wound using a mechanism called restitution. In the colon, where there are no villi, restitution is particularly rapid. Following restitution, tight junctions have to be re-sealed to re-establish barrier function, which occurs rapidly by recycling intact tight junction proteins. The intestinal barrier is further disrupted during injury by inflammation, including transmigration of neutrophils that disrupt tight junctions. An additional critical element of barrier function is the continuous renewal of epithelium via proliferation of stem cells contained within the intestinal crypts. Proliferation increases following injury to replace lost epithelium. Collectively, barrier function includes mechanisms to maintain a healthy epithelial barrier, repair in the acute phases of injury and inflammation, and replace epithelium via stem cell activity.

Simple Summary Equine colic places a substantial financial burden on horse owners and the equine industry each year. Equine veterinary research is focused on preventing colic on the farm whenever possible and improving treatment options available to veterinarians in the field and referral hospitals. It is important for scientists to have a detailed understanding of the intestinal damage created by different types of colic in foals and adult horses so they can better target certain cell types or tissue systems when investigating new treatment options. This review article summarizes recent works in the field of intestinal injury research and describes the potential roles of various intestinal systems, such as the enteric nervous system (ENS), in repairing the intestine after colic injury and how these systems mature in early life. Abstract Colic is a leading cause of death in horses, with the most fatal form being strangulating obstruction which directly damages the intestinal barrier. Following surgical intervention, it is imperative that the intestinal barrier rapidly repairs to prevent translocation of gut bacteria and their products and ensure survival of the patient. Age-related disparities in survival have been noted in many species, including horses, humans, and pigs, with younger patients suffering poorer clinical outcomes. Maintenance and repair of the intestinal barrier is regulated by a complex mucosal microenvironment, of which the ENS, and particularly a developing network of subepithelial enteric glial cells, may be of particular importance in neonates with colic. Postnatal development of an immature enteric glial cell network is thought to be driven by the microbial colonization of the gut and therefore modulated by diet-influenced changes in bacterial populations early in life. Here, we review the current understanding of the roles of the gut microbiome, nutrition, stress, and the ENS in maturation of intestinal repair mechanisms after foaling and how this may influence age-dependent outcomes in equine colic cases.

Postoperative ileus (POI), a decrease in gastrointestinal motility after surgery, is an important problem facing human and veterinary patients. Retrospective study indicates that 37.5% of horses that develop POI following small intestinal resection will not survive to discharge. The two major components of POI pathophysiology are a neurogenic phase which is then propagated by an inflammatory phase. Perioperative care has also been implicated, namely the use of opioid therapy, inappropriate fluid therapy and electrolyte imbalances. Current therapy for POI variably includes an early return to feeding to induce physiological gastrointestinal motility, reducing the inflammatory response with agents such as non-steroidal anti-inflammatory drugs, and use of prokinetic therapy such as lidocaine. However, optimal management of POI remains controversial. A better understanding of the roles of the gastrointestinal microbiota, intestinal mucosal barrier function, the post-surgical inflammatory response, as well as enteric glial cells, a component of the enteric nervous system, in modulating postoperative motility and the pathogenesis of POI may provide future targets for prevention and/or therapy of POI.

Management factors associated with colic, particularly related to stall confinement and nutrition, have been linked to alterations in gastrointestinal mucosal transport, motility, and microbiome, which in turn creates conditions that induce colic. In particular, meal feeding creates large changes in water movement in and out of the colon and alters the microbiome. These conditions may in turn result in colic conditions such as large colon impaction or large colon volvulus. In addition, a range of management and nutritional factors have been found to place horses at risk of select colic conditions such as ileal impaction. Other specific colic conditions, such as strangulating lipomas, may be related to fat metabolism in geldings and ponies, although the association with nutrition and the endocrine system are less well defined. It has long been understood that parasites are associated with colic, and with the advent of highly effective anthelmintics, parasite-induced colic has been markedly reduced. Nonetheless, equine mangers and veterinarians have to be aware of changes in parasite resistance or patterns of activity, such as the resurgence of large strongyles with surveillance-based management of parasites. Overall, understanding management risk factors can lead to recommendations that prevent colic in horses. Additional study of these factors may ultimately lead to reductions in the prevalence of colic by suggesting optimal management practices.

Background Equine coronavirus (ECoV) is an emerging pathogen associated with fever and enteric disease in adult horses. Clinical features of ECoV infection have been described, but no study has compared these features to those of Salmonella infections. Objectives Compare the clinical features of ECoV infection with enteric salmonellosis and establish a disease signature to increase clinical suspicion of ECoV infection in adult horses. Animals Forty-three horses >1 year of age with results of CBC, serum biochemistry, and fecal diagnostic testing for ECoV and Salmonella spp. Methods Medical records of horses presented to the North Carolina State University Equine and Farm Animal Veterinary Center (2003-016) were retrospectively reviewed. Horses were divided into 3 groups based on fecal diagnostic test results: ECoV-positive, Salmonella-positive, or unknown diagnosis (UNK). Time of year presented, clinical signs, CBC, and serum biochemistry test results were recorded. Data were analyzed by 1-way analysis of variance, Kruskal-Wallis test, or Fisher's exact test with significance set at P Results Most common presenting complaints were fever and colic and were similar across groups. Horses with ECoV had significantly decreased neutrophil counts when compared to those with no diagnosis but were not different from horses with Salmonella. Horses with Salmonella had significantly lower mean leukocyte counts compared to those with UNK. No significant differences were found among groups for any other examined variable. Conclusions and clinical importance Equine coronavirus and Salmonella infections share clinical features, suggesting both diseases should be differential diagnoses for horses with fever and enteric clinical signs.

Disruptions in the intestinal epithelial barrier can result in devastating consequences and a multitude of disease syndromes, particularly among preterm neonates. The association between barrier dysfunction and intestinal dysbiosis suggests that the intestinal barrier function is interactive with specific gut commensals and pathogenic microbes. In vitro and in vivo studies demonstrate that probiotic supplementation promotes significant upregulation and relocalization of interepithelial tight junction proteins, which form the microscopic scaffolds of the intestinal barrier. Probiotics facilitate some of these effects through the ligand-mediated stimulation of several toll-like receptors that are expressed by the intestinal epithelium. In particular, bacterial-mediated stimulation of toll-like receptor-2 modulates the expression and localization of specific protein constituents of intestinal tight junctions. Given that ingested prebiotics are robust modulators of the intestinal microbiota, prebiotic supplementation has been similarly investigated as a potential, indirect mechanism of barrier preservation. Emerging evidence suggests that prebiotics may additionally exert a direct effect on intestinal barrier function through mechanisms independent of the gut microbiota. In this review, we summarize current views on the effects of pro- and prebiotics on the intestinal epithelial barrier as well as on non-epithelial cell barrier constituents, such as the enteric glial cell network. Through continued investigation of these bioactive compounds, we can maximize their therapeutic potential for preventing and treating gastrointestinal diseases associated with impaired intestinal barrier function and dysbiosis.

Larazotide acetate (LA) is a single-chain peptide of eight amino acids that acts as a tight junction regulator to restore intestinal barrier function. LA is currently being studied in phase III clinical trials and is orally administered to adult patients with celiac disease as an adjunct therapeutic to enhance intestinal barrier function that has been disrupted by gliadin-induced immune reactivity. Mechanistically, LA is thought to act as a zonulin antagonist to reduce zonulin-induced increases in barrier permeability and has been associated with the redistribution and rearrangement of tight junction proteins and actin filaments to restore intestinal barrier function. More recently, LA has been linked to inhibition of myosin light chain kinase, which likely reduces tension on actin filaments, thereby facilitating tight junction closure. Small (rodent) and large (porcine) animal studies have been conducted that demonstrate the importance of LA as a tight junction regulatory peptide in conditions other than celiac disease, including collagen-induced arthritis in mice and intestinal ischemic injury in pigs.

Eosinophilic esophagitis (EoE) is a chronic atopic disease that has become increasingly prevalent over the past 20 years. A first-line pharmacologic option is topical/swallowed corticosteroids, but these are adapted from asthma preparations such as fluticasone from an inhaler and yield suboptimal response rates. There are no FDA-approved medications for the treatment of EoE, and esophageal-specific drug formulations are lacking. We report the development of two novel esophageal-specific drug delivery platforms. The first is a fluticasone-eluting string that could be swallowed similar to the string test “entero-test” and used for overnight treatment, allowing for a rapid release along the entire length of esophagus. In vitro drug release studies showed a target release of 1 mg/day of fluticasone. In vivo pharmacokinetic studies were carried out after deploying the string in a porcine model, and our results showed a high local level of fluticasone in esophageal tissue persisting over 1 and 3 days, and a minimal systemic absorption in plasma. The second device is a fluticasone-eluting 3D printed ring for local and sustained release of fluticasone in the esophagus. We designed and fabricated biocompatible fluticasone-loaded rings using a top-down, Digital Light Processing (DLP) Gizmo 3D printer. We explored various strategies of drug loading into 3D printed rings, involving incorporation of drug during the print process (pre-loading) or after printing (post-loading). In vitro drug release studies of fluticasone-loaded rings (pre and post-loaded) showed that fluticasone elutes at a constant rate over a period of one month. Ex vivo pharmacokinetic studies in the porcine model also showed high tissue levels of fluticasone and both rings and strings were successfully deployed into the porcine esophagus in vivo. Given these preliminary proof-of-concept data, these devices now merit study in animal models of disease and ultimately subsequent translation to testing in humans.

Intestinal ischemia results in mucosal injury, including paracellular barrier loss due to disruption of tight junctions. Larazotide acetate (LA), a small peptide studied in Phase III clinical trials for treatment of celiac disease, regulates tight junctions (TJs). We hypothesized that LA would dose-dependently hasten recovery of intestinal ischemic injury via modulation of TJs. Ischemia-injured tissue from 6-8-week-old pigs was recovered in Ussing chambers for 240-minutes in the presence of LA. LA (1 μM but not 0.1 μM or 10 μM) significantly enhanced transepithelial electrical resistance (TER) above ischemic injured controls and significantly reduced serosal-to-mucosal flux LPS (P.05). LA (1 μM) enhanced localization of the sealing tight junction protein claudin-4 in repairing epithelium. To assess for the possibility of fragmentation of LA, anin vitroenzyme degradation assay using the brush border enzyme aminopeptidase M, revealed generation of peptide fragments. Western blot analysis of total protein isolated from uninjured and ischemia-injured porcine intestine showed aminopeptidase M enzyme presence in both tissue types, and mass spectrometry analysis of samples collected duringex vivoanalysis confirmed formation of LA fragments. Treatment of tissues with LA fragments had no effect alone, but treatment with a fragment missing both amino-terminus glycines inhibited barrier recovery stimulated by 1 μM LA. To reduce potential LA inhibition by fragments, a D-amino acid analog of larazotide Analog #6, resulted in a significant recovery response at a 10-fold lower dose (0.1 μM) similar in magnitude to that of 1 μM LA. We conclude that LA stimulates repair of ischemic-injured epithelium at the level of the tight junctions, at an optimal dose of 1 μM LA. Higher doses were less effective because of inhibition by LA fragments, which could be subverted by chirally-modifying the molecule, or microdosing LA.

There is no FDA approved therapy for the treatment of celiac disease (CeD), aside from avoidance of dietary gluten. Larazotide acetate (LA) is a first in class oral peptide developed as a tight junction regulator, which is a lead candidate for management of CeD. A delayed release formulation was tested in vitro and predicted release in the mid duodenum and jejunum, the target site of CeD. The aim of this study was to follow the concentration versus time profile of orally administered LA in the small intestine using a porcine model. A sensitive liquid chromatography/tandem mass spectrometry method was developed to quantify LA concentrations in porcine intestinal fluid samples. Oral dosing of LA (1 mg total) in overnight fasted pigs resulted in time dependent appearance of LA in the distal duodenum and proximal jejunum. Peak LA concentrations (0.32–1.76 μM) occurred at 1 hour in the duodenum and in proximal jejunum following oral dosing, with the continued presence of LA (0.02–0.47 μM) in the distal duodenum and in proximal jejunum (0.00–0.43 μM) from 2 to 4 hours following oral dosing. The data shows that LA is available in detectable concentrations at the site of CeD.

The pig is a powerful model for intestinal barrier studies, and it is important to carefully plan animal care and handling for optimal study design as psychological and physiological stressors significantly impact intestinal mucosal barrier function. Here, we report the effects of a period of environmental acclimation versus acute transport stress on mucosal barrier repair after intestinal ischemic injury. Jejunal ischemia was induced in young pigs which had been allowed to acclimate to a biomedical research housing environment or had been transported immediately prior to experimental injury (non-acclimated). Mucosa was then incubated ex vivo on Ussing chambers. In uninjured mucosa, there was no difference in transepithelial electrical resistance (TEER) or epithelial integrity between groups. However, acclimated pigs had increased macromolecular flux as compared to non-acclimated pigs during the first hour of ex vivo incubation. Ischemia induced greater epithelial loss in non-acclimated pigs as compared to acclimated pigs, yet this group achieved greater wound healing during recovery. Non-acclimated pigs had more robust TEER recovery ex vivo following injury versus acclimated pigs. The expression pattern of the tight junction protein claudin-4 was disrupted in acclimated pigs following recovery but showed enhanced localization to the apical membrane in non-acclimated pigs following recovery. Acute transport stress increases mucosal susceptibility to epithelial loss but also primes the tissue for a more robust barrier repair response. Alternatively, environmental acclimation increases leak pathway and diminishes barrier repair responses after ischemic injury.

The intestinal epithelial apical junctional complex, which includes tight and adherens junctions, contributes to the intestinal barrier function via their role in regulating paracellular permeability. Myosin light chain II (MLC-2), has been shown to be a critical regulatory protein in altering paracellular permeability during gastrointestinal disorders. Previous studies have demonstrated that phosphorylation of MLC-2 is a biochemical marker for perijunctional actomyosin ring contraction, which increases paracellular permeability by regulating the apical junctional complex. The phosphorylation of MLC-2 is dominantly regulated by myosin light chain kinase- (MLCK-) and Rho-associated coiled-coil containing protein kinase- (ROCK-) mediated pathways. In this review, we aim to summarize the current state of knowledge regarding the role of MLCK- and ROCK-mediated pathways in the regulation of the intestinal barrier during normal homeostasis and digestive diseases. Additionally, we will also suggest potential therapeutic targeting of MLCK- and ROCK-associated pathways in gastrointestinal disorders that compromise the intestinal barrier.

Esophageal injury from acid exposure related to gastroesophageal reflux disease is a common problem and a risk factor for development of Barrett’s esophagus and esophageal adenocarcinoma. Our previous work highlights the benefits of using porcine esophagus to study human esophageal disease because of the similarities between porcine and human esophagus. In particular, esophageal submucosal glands (ESMGs) are present in human esophagus and proximal porcine esophagus but not in rodent esophagus. Although CFTR is expressed in the ducts of ESMGs, very little is known about CFTR and alternate anion channels, including ClC-2, in the setting of acid-related esophageal injury. After finding evidence of CFTR and ClC-2 in the basal layers of the squamous epithelium, and in the ducts of the ESMGs, we developed an ex vivo porcine model of esophageal acid injury. In this model, esophageal tissue was placed in Ussing chambers to determine the effect of pretreatment with the ClC-2 agonist lubiprostone on tissue damage related to acid exposure. Pretreatment with lubiprostone significantly reduced the level of acid injury and significantly augmented the recovery of the injured tissue ( P < 0.05). Evaluation of the interepithelial tight junctions showed well-defined membrane localization of occludin in lubiprostone-treated injured tissues. Pretreatment of tissues with the Na+-K+-2Cl−cotransporter inhibitor bumetanide blocked lubiprostone-induced increases in short-circuit current and inhibited the reparative effect of lubiprostone. Furthermore, inhibition of ClC-2 with ZnCl2blocked the effects of lubiprostone. We conclude that ClC-2 contributes to esophageal protection from acid exposure, potentially offering a new therapeutic target.NEW & NOTEWORTHY This research is the first to describe the presence of anion channels ClC-2 and CFTR localized to the basal epithelia of porcine esophageal mucosa and the esophageal submucosal glands. In the setting of ex vivo acid exposure, the ClC-2 agonist lubiprostone reduced acid-related injury and enhanced recovery of the epithelial barrier. This work may ultimately provide an alternate mechanism for treating gastroesophageal reflux disease.

The intestinal epithelial monolayer forms a transcellular and paracellular barrier that separates luminal contents from the interstitium. The paracellular barrier consists of a highly organized complex of intercellular junctions that is primarily regulated by apical tight junction proteins and tight junction-associated proteins. This homeostatic barrier can be lost through a multitude of injurious events that cause the disruption of the tight junction complex. Acute repair after injury leading to the reestablishment of the tight junction barrier is crucial for the return of both barrier function as well as other cellular functions, including water regulation and nutrient absorption. This review provides an overview of the tight junction complex components and how they link to other plasmalemmal proteins, such as ion channels and transporters, to induce tight junction closure during repair of acute injury. Understanding the components of interepithelial tight junctions and the mechanisms of tight junction regulation after injury is crucial for developing future therapeutic targets for patients experiencing dysregulated intestinal permeability.

BACKGROUND Continuous digital cryotherapy experimentally prevents development and reduces severity of sepsis-associated laminitis. A sleeve style ice boot where ice is in direct contact with the skin, and water drains from the boot is being used clinically for distal limb cryotherapy. The degree of cooling achieved by this boot is unknown. OBJECTIVES Evaluate skin and lamellar cooling after application of the ice sleeve in healthy horses, and the same horses during an endotoxaemia model. STUDY DESIGN Prospective study, crossover design. METHODS In eight healthy horses thermocouples were inserted into dorsal lamellae of both front feet, and under skin on both metacarpi. One forelimb received cryotherapy using sleeve style ice boot, with contralateral limb as control. Temperature was recorded on data logging devices at 5 min intervals during each cryotherapy session. Day 1: temperature data was collected for healthy horses. Day 2: data was collected for the same horses during i.v. administration of endotoxin. RESULTS In healthy and endotoxaemic horses, the sleeve style ice boot significantly decreased mean skin (7.2°C and 5.8°C respectively) and lamellar (10.8°C and 9.6°C respectively) temperatures compared with control limbs (P

Background Sucralfate is a gastroprotectant with no known systemic effects. The efficacy of sucralfate for prevention and treatment of stress‐related mucosal diseases (SRMD) in dogs is unknown. Hypothesis/Objectives To develop a canine ex vivo model of SRMD and to determine the effect of sucralfate on mucosal barrier function in this model. Animals Gastric antral mucosa was collected immediately postmortem from 29 random‐source apparently healthy dogs euthanized at a local animal control facility. Methods Randomized experimental trial. Sucralfate (100 mg/mL) was applied to ex vivo canine gastric mucosa concurrent with and after acid injury. Barrier function was assessed by measurement of transepithelial electrical resistance (TER) and radiolabeled mannitol flux. Results Application of acidified Ringers solution to the mucosal side of gastric antrum caused a reduction in gastric barrier function, and washout of acidified Ringers solution allowed recovery of barrier function (TER: 34.0 ± 2.8% of control at maximum injury, 71.3 ± 5.5% at recovery, P

The equine intestinal mucosa is intimately involved in maintaining homeostasis both on a systemic level by controlling extracellular fluid movement and at the local level to maintain barrier function. Horses are particularly susceptible to the clinical syndrome of colic, with the most severe cases involving strangulating obstruction that induces ischemia. Because of the mucosal vascular architecture, the mucosal epithelium is particularly susceptible to ischemic injury. The potential for reperfusion injury has been investigated and found to play a minimal role. However, inflammation does affect mucosal repair. Mechanisms of repair, including villus contraction, epithelial restitution, and tight junction closure, are critical to reforming the mucosal barrier. Nonsteroidal anti-inflammatory drugs have an impact on this repair, particularly at the level of the tight junctions. Completion of mucosal regeneration requires proliferation, which is now being actively studied in equine enteroids. All of these aspects of equine mucosal pathobiology are reviewed in depth.

OBJECTIVES/GOALS: An age-dependent restitution defect in our neonatal pig intestinal ischemia model is rescued by unknown factors within homogenized mucosa of weaned pigs. A postnatally maturing network of enteric glia regulates the epithelial barrier, so we aim to show rescue is due to replacement of glial factors. METHODS/STUDY POPULATION: Jejunal tissues from suckling or weaned pigs were assessed by RNAseq and processed for immunofluorescent histology and 3-D volume imaging. Jejunal ischemia was surgically induced in weaned pigs and injured mucosa was recovered ex vivo with or without the glial inhibitor fluoroacetate (FA) while monitoring transepithelial electrical resistance (TER). RESULTS/ANTICIPATED RESULTS: Ingenuity Pathways Analysis of RNAseq data revealed significant suppression of numerous pathways critical for epithelial wound healing in suckling pigs (Z-score in vitro to further define postnatal development of barrier repair.

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 injury, although typically associated with ischemic lesions, occurs during any obstructive intestinal disease to varying degrees depending on the type of obstruction and the extent of vascular compromise. In addition, in some instances, the intestinal lumen is patent, but there is vascular compromise—for example, in nonstrangulating infarctions. Intestinal obstructive lesions are classified as either simple or strangulating obstructions. A great deal of work has been done to assess the level of injury encountered with these lesions at surgery and during the postsurgical phase following correction and subsequent reperfusion of these lesions. Although more is known about mucosal injury than about injury encountered in other intestinal layers, it is clear that substantial injury also occurs at the levels of the serosa and the muscularis, which very likely contributes to postoperative complications such as adhesion formation and ileus. Understanding the pathophysiology of these lesions allows the surgeon to more adequately perform surgical and postoperative procedures to optimize patient survival. For example, attention has been focused on the development of intestinal injury during reperfusion, and the possibility of inhibiting these lesions with various treatments. Furthermore, to combat the postoperative complication of serosal inflammation and secondary adhesion formation, surgeons have adopted a number of new treatments.

Gastrointestinal disease is a prevalent cause of morbidity and mortality and the use of animal models have been instrumental in studying mechanisms of digestive pathophysiology. As investigators attempt to translate the wealth of basic science information developed from rodent models, large animal models provide a number of translational advantages. The pig, in particular, is arguably one of the most powerful models of human organ systems, including the gastrointestinal tract. The pig has provided important tools and insight into intestinal ischemia/reperfusion injury, intestinal mucosal repair, as well as new insights into esophageal injury and repair. Porcine model development has taken advantage of the size of the animal, allowing increased surgical and endoscopic access. In addition, cellular tools such as the intestinal porcine epithelial cell (IPEC-J2) line and porcine enteroids are providing the methodology to translate basic science findings using in-depth mechanistic analyses. Further opportunities in porcine digestive disease modeling include developing additional transgenic pig strains. Collectively, porcine models hold great promise for the future of clinically relevant digestive disease research. Keywords: Pig, Ischemia/Reperfusion Injury, Mucosal Repair, Tight Junction

REASONS FOR PERFORMING STUDY The large size of the adult horse prevents the use of advanced imaging modalities in most areas of the axial skeleton, including the lumbosacral vertebral column. Traditional imaging techniques are frequently unable to pinpoint the underlying pathology in horses with caudal back pain. In man, lumbosacral epiduroscopy is used to diagnose and treat subjects with chronic back and leg pain. This technique may close the diagnostic gap in horses with similar clinical signs. OBJECTIVES To evaluate the safety and feasibility of lumbosacral epiduroscopy in the standing adult horse. STUDY DESIGN Descriptive, experimental study. METHODS Seven adult horses weighing 504-578 kg were sedated and restrained in stocks in preparation for aseptic surgery. Vascular dilators of increasing size were inserted cranial to the first moveable vertebra caudal to the sacrum to facilitate a minimally invasive approach into the epidural space. A flexible video-endoscope was introduced and advanced as far as its 60-cm working length permitted. Pre-, intra- and post-operative plasma cortisol samples were collected, and neurological and lameness examinations were performed prior to and during the 2 weeks following the procedure. Post-mortem examinations were conducted in 5 of the 7 horses. RESULTS Standing lumbosacral epiduroscopy was well tolerated by all horses. The anatomic structures in the epidural space (dura mater, spinal nerve roots, fat and blood vessels) were followed as far cranial as the thoracolumbar region. No complications related to the procedure were noted in the 2-week monitoring period following epiduroscopy. Small, organised haematomas were identified in the sacral epidural space during necropsy in one horse. No abnormalities were seen in the other 4 animals. CONCLUSIONS Lumbosacral epiduroscopy can be performed safely in sedated standing horses. The procedure may become a valuable diagnostic tool in horses with caudal back or hindlimb pain of unknown origin.

REASONS FOR PERFORMING STUDY Clinically useful biomarkers are needed for early identification of endotoxaemic horses. Soluble CD14 (sCD14) is amplified early in response to inflammatory signals, including bacterial lipopolysaccharide (LPS), and may prove a useful biomarker for clinical endotoxaemia. OBJECTIVES The aim of this study was to determine if sCD14 could serve as a more reliable biomarker of the clinical signs of endotoxaemia, compared to measuring LPS alone. STUDY DESIGN Prospective observational study in horses at a veterinary teaching hospital. METHODS Plasma samples were collected from 20 healthy horses and 35 horses presenting for emergency evaluation. Horses were classified as clinically endotoxaemic, using previously established criteria, if they had a heart rate >70 beats/min, packed cell volume >45% and/or a lesion likely to result in endotoxaemia. Soluble CD14 was measured using a cytometric bead-based assay and LPS was measured using a Limulus amoebocyte lysate (LAL) assay. RESULTS Soluble CD14 was higher in horses classified as clinically endotoxaemic (median 1102 ng/ml, interquartile range 439 ng/ml), compared to clinically nonendotoxaemic (median 692 ng/ml, interquartile range 455 ng/ml, P = 0.03. There was no difference in LPS concentrations between clinically nonendotoxaemic (median 5.4 endotoxin units [EU]/ml, interquartile range 5 EU/ml) and endotoxaemic horses (median 7.2 EU/ml, interquartile range 17 EU/ml, P = 0.2). There was no correlation between sCD14 and LPS values in paired serum samples. LPS and sCD14 values were used to generate a receiver operating characteristic curve. The area under the curve for LPS and sCD14 was