Protocol to culture enteric glial cells from the submucosal and myenteric plexi of neonatal and juvenile pig colons.

Here, we present our protocol to culture enteric glial cells from the submucosal and myenteric plexus of neonatal and juvenile pig colons. We describe steps for colon isolation, microdissection, and enzymatic and mechanical dissociation. We include procedures for passaging and analyzing cell yield, freeze/thaw efficiency, and purity. This protocol allows for the generation of primary cultures of enteric glial cells from single-cell suspensions of microdissected layers of the colon wall and can be used to culture enteric glia from human colon specimens. For complete details on the use and execution of this protocol, please refer to Ziegler et al.1.

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 (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.

Hypoxia Primes Human ISCs for Interleukin-Dependent Rescue of Stem Cell Activity.

BACKGROUND AND AIMS: Hypoxia in the intestinal epithelium can be caused by acute ischemic events or chronic inflammation in which immune cell infiltration produces inflammatory hypoxia starving the mucosa of oxygen. The epithelium has the capacity to regenerate after some ischemic and inflammatory conditions suggesting that intestinal stem cells (ISCs) are highly tolerant to acute and chronic hypoxia; however, the impact of hypoxia on human ISC (hISC) function has not been reported. Here we present a new microphysiological system (MPS) to investigate how hypoxia affects hISCs from healthy donors and test the hypothesis that prolonged hypoxia modulates how hISCs respond to inflammation-associated interleukins (ILs). METHODS: hISCs were exposed to <1.0% oxygen in the MPS for 6, 24, 48, and 72 hours. Viability, hypoxia-inducible factor 1a (HIF1a) response, transcriptomics, cell cycle dynamics, and response to cytokines were evaluated in hISCs under hypoxia. HIF stabilizers and inhibitors were screened to evaluate HIF-dependent responses. RESULTS: The MPS enables precise, real-time control and monitoring of oxygen levels at the cell surface. Under hypoxia, hISCs maintain viability until 72 hours and exhibit peak HIF1a at 24 hours. hISC activity was reduced at 24 hours but recovered at 48 hours. Hypoxia induced increases in the proportion of hISCs in G1 and expression changes in 16 IL receptors. Prolyl hydroxylase inhibition failed to reproduce hypoxia-dependent IL-receptor expression patterns. hISC activity increased when treated IL1ß, IL2, IL4, IL6, IL10, IL13, and IL25 and rescued hISC activity caused by 24 hours of hypoxia. CONCLUSIONS: Hypoxia pushes hISCs into a dormant but reversible proliferative state and primes hISCs to respond to a subset of ILs that preserves hISC activity. These findings have important implications for understanding intestinal epithelial regeneration mechanisms caused by inflammatory hypoxia.

A novel case of tracheal injury secondary to gunshot trauma in a white rhinoceros (Ceratotherium simum).

OBJECTIVE: To describe a case of tracheal injury secondary to gunshot trauma in a rhinoceros. ANIMALS: 5-year-old female white rhinoceros (Ceratotherium simum). CLINICAL PRESENTATION, PROGRESSION, AND PROCEDURES: The rhinoceros was found alive with an apparent bullet entry wound cranial to the left shoulder. The rhinoceros was agitated and had bilateral epistaxis and increased respiratory noise. Immobilization of the animal facilitated closer examination and initiation of medical therapy. Radiographs obtained of the neck region at this first examination were nondiagnostic. Subsequent immobilization events allowed for further diagnostics and treatment. TREATMENT AND OUTCOME: Initial treatment included a broad-spectrum antibiotic and a corticosteroid. Five days following the injury, the rhinoceros was considered stable, and the animal was immobilized to investigate the cause of the epistaxis and respiratory signs. Tracheoscopy revealed a full-thickness penetrating wound in the mid to caudal region of the trachea, and the surface of a metallic projectile was viewed within the wound. Medical treatment was continued and the rhinoceros was managed conservatively. At 14 days, radiographs of the neck made with a more powerful unit revealed tissue emphysema dorsal to the trachea. A subsequent tracheoscopy 54 days after injury revealed a granulated wound. Follow-up at 4 years after injury determined that the rhinoceros was reported to be behaving normally and had successfully calved. CLINICAL RELEVANCE: Gunshot wounds associated with poaching are a prevalent problem in rhinoceros in Africa. Although more aggressive therapy including surgery may likely be considered in zoo or domestic animals, limited conservative treatment was successful in this wild-managed rhinoceros.

Risk factors associated with an outbreak of equine coronavirus at a large farm in North Carolina.

Background: Equine coronavirus (ECoV) leads to outbreaks with variable morbidity and mortality. Few previous reports of risk factors for infection are available in the literature. Objectives: To describe unique clinical findings and risk factors for infection and development of clinical disease. Animals: 135 horses on a farm affected by ECoV outbreak. Methods: Retrospective cohort study. Data obtained included age, breed, gender, activity level, housing, and feed at the onset of the outbreak. Factors were evaluated for assessment of risk of infection using simple logistic regression or Fisher's exact test. Significance was set at p ≤ 0.05. Results and findings: Forty-three of 54 (79.6%) horses tested on the farm were positive on fecal PCR for ECoV, and 17 horses (12.6%) developed clinical signs consistent with ECoV. Out of 17 horses in which the presence or absence of signs of colic was noted, 6 of 17 (35.3%) showed signs of colic. Three of these horses had small colon impactions, 2 of which required surgical intervention. Significant risk factors for having positive PCR results included being primarily stalled (OR 167.1, 95% CI 26.4-1719), housing next to a positive horse (OR 7.5, 95% CI 3.1-19.0), being in work (OR 26.9, 95% CI 4.6-281.9), being fed rationed hay vs. ad libitum (OR 1,558, 95% CI 130.8-15,593), and being fed alfalfa hay (OR 1,558, 95% CI 130.8-15,593). Conclusions and clinical importance: This report describes risk factors for ECoV infection many of which were associated with intensive management of show horses. Clinicians should be aware that clinical signs vary and can include severe colic.

A new microphysiological system shows hypoxia primes human ISCs for interleukin-dependent rescue of stem cell activity.

Background & Aims: Hypoxia 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. Methods: hISCs were exposed to <1.0% oxygen in the MPS for 6-, 24-, 48- & 72hrs. Viability, HIF1α response, transcriptomics, cell cycle dynamics, and hISC response to cytokines were evaluated. Results: The 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 interleukins. Conclusions: Hypoxia 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.

Mechanisms and modeling of wound repair in the intestinal epithelium.

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.

Multi-Institutional Retrospective Case-Control Study Evaluating Clinical Outcomes of Foals with Small Intestinal Strangulating Obstruction: 2000-2020.

Lower survival has been reported in foals than adults with small intestinal strangulating obstruction (SISO), but age-dependent outcomes have not been examined directly. Hospital records were collected from five US academic referral hospitals. It was hypothesized that foals would exhibit lower survival than case-matched adults. Foal cases 6-months-of-age or younger, and adult cases between 2- and 20-years-of-age were collected. Data revealed 24 of 25 (96.0%) foals and 66 of 75 (88.0%) adults that were recovered from surgery for SISO survived to hospital discharge. Sixteen of the total 41 (39.0%) foals studied were euthanized intraoperatively, whereas 30 of 105 (28.6%) adults were euthanized intraoperatively. Common lesions in foals that were recovered from surgery were volvulus (n = 13) and intussusception (n = 5), whereas common lesions in adults were volvulus (n = 25) and strangulating lipoma (n = 23). This study was limited by incomplete medical records, relatively small sample size, and lack of long-term follow-up. Unexpectedly, short-term survival tended to be higher in foals than adults and may have been partly driven by case selection prior to referral or surgery or decision-making intraoperatively. More optimism toward surgical treatment of foals with SISO may be warranted.

Porcine Models of the Intestinal Microbiota: The Translational Key to Understanding How Gut Commensals Contribute to Gastrointestinal Disease.

In the United States, gastrointestinal disorders account for in excess of $130 billion in healthcare expenditures and 22 million hospitalizations annually. Many of these disorders, including necrotizing enterocolitis of infants, obesity, diarrhea, and inflammatory bowel disease, are associated with disturbances in the gastrointestinal microbial composition and metabolic activity. To further elucidate the pathogenesis of these disease syndromes as well as uncover novel therapies and preventative measures, gastrointestinal researchers should consider the pig as a powerful, translational model of the gastrointestinal microbiota. This is because pigs and humans share striking similarities in their intestinal microbiota as well as gastrointestinal anatomy and physiology. The introduction of gnotobiotic pigs, particularly human-microbial associated pigs, has already amplified our understanding of many gastrointestinal diseases that have detrimental effects on human health worldwide. Continued utilization of these models will undoubtedly inform translational advancements in future gastrointestinal research and potential therapeutics.

Non-steroidal anti-inflammatory drugs in equine orthopaedics.

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.

Age-Dependent Intestinal Repair: Implications for Foals with Severe Colic.

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: Comparative Pathophysiology and Future Therapies.

Postoperative ileus (POI), a decrease in gastrointestinal motility after surgery, is an important problem facing human and veterinary patients. 37.5% of horses that develop POI following small intestinal (SI) 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 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 motility, reducing the inflammatory response with agents such as non-steroidal anti-inflammatory drugs (NSAIDs), and use of prokinetic therapy such as lidocaine. However, optimal management of POI remains controversial. Further understanding of the roles of the gastrointestinal microbiota, intestinal barrier function, the post-surgical inflammatory response, as well as enteric glial cells, a component of the enteric nervous system, in modulating postoperative gastrointestinal motility and the pathogenesis of POI may provide future targets for prevention and/or therapy of POI.

Probiotics, Prebiotics and Epithelial Tight Junctions: A Promising Approach to Modulate Intestinal Barrier Function.

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 induces recovery of ischemia-injured porcine jejunum via repair of tight junctions.

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<0.05). 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, an in vitro enzyme 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 during ex vivo analysis 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.

Larazotide acetate: a pharmacological peptide approach to tight junction regulation.

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.

In vivo assessment of a delayed release formulation of larazotide acetate indicated for celiac disease using a porcine model.

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.

Environmental stressors affect intestinal permeability and repair responses in a pig intestinal ischemia model.

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.

Comparison of histomorphometric characteristics of dorsal colon and pelvic flexure biopsy specimens obtained from horses with large colon volvulus that underwent resection.

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.

The Regulation of Intestinal Mucosal Barrier by Myosin Light Chain Kinase/Rho Kinases.

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.

Lubiprostone protects esophageal mucosa from acid injury in porcine esophagus.

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 ZnCl2 blocked 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.