About the lab
Led by Drs. Anthony Blikslager and Amanda Ziegler, the Comparative Gastroenterology Lab aims to study translational development of gut health using large animal models, primarily pigs. In multidisciplinary collaboration with Drs. Jack Odle, Laurianne Van Landeghem and Scott Magness, the lab is USDA and NIH funded to study intensively the roles of nutritional inputs in the development of a healthy and disease-resistant intestinal microbiome, enteric nervous system, and epithelial barrier. Our ultimate objective is to develop clinical interventions to aid in restoring the mucosal barrier in patients suffering from diseases associated with increased intestinal permeability.
Featured projects (1)
This work centers on the use of large animal models to study mechanisms of intestinal epithelial barrier repair following ischemic injury. I recently identified a defect in epithelial restitution in our neonatal pig model and I am exploring the development of intestinal mucosal repair mechanisms during the postnatal period in pigs. My current work is focused on the maturation of the enteric glial network in the neonatal and juvenile aged pigs and its role of in signaling for epithelial barrier repair. These findings are highly translatable to human infants and neonatal equine patients suffering from diseases involving intestinal ischemia such as necrotizing enterocolitis and voluvulus. My long-term research goals are to identify and rescue mechanisms of barrier repair in neonates to facilitate development of specific clinical intervention strategies in neonatal patients with intestinal disease characterized by defective epithelial barrier function.
Featured research (5)
In our pig intestinal ischemia model, a neonatal defect in epithelial barrier restitution in the jejunum can be rescued by ischemic mucosal homogenate from weaned pigs. This is associated with an immature enteric glial cell (EGC) network, a known driver of restitution that matures postnatally partly in response to microbial colonization, which can be modulated with dietary prebiotic fiber. Therefore, we hypothesized that dietary oligosaccharide supplementation accelerates postnatal microbial colonization and EGC network maturation in the jejunum and colon, thus enhancing restitution after ischemic injury. After 24-hours of colostrum, piglets were fed control or oligosaccharide-supplemented formula (control-fed or prebiotic-fed) for 21 days and fecal swabs were sequenced for 16S rDNA. Intestinal samples were collected for western blot, imaging, and EGC culture. Surgically ischemia-injured jejunal and colonic mucosal samples from select day 14 pigs were recovered ex vivo while monitoring epithelial barrier function by transepithelial electrical resistance (TEER). Migration abilities, calcium responses to ATP, paracrine effects on IPEC-J2 cell restitution, and protein secretome were assessed in jejunal and colonic EGC cultures. Colonic microbial taxa changed in a time- and diet-dependent manner with the prebiotic-fed taxa clustering by day 7 and becoming progressively more tightly clustered over time (P<.050). TEER and histology of uninjured jejunum and colon were unaffected by diet. Following ischemia, low initial TEER recovered to control levels in the control-fed colon but not prebiotic-fed colon (diet and injury interaction, P=.038), while diet had no effect on jejunal TEER recovery. Prebiotic-fed colon had lower levels of the EGC markers glial fibrillary acidic protein (GFAP) and S-100B at day 21 (P<.050), and subjectively reduced density of GFAP+ and S-100B+ EGC were noted in preliminary volume imaging of prebiotic-fed jejunal submucosa at days 14 and 21. EGC from prebiotic-fed colonic submucosa showed decreased chemotactic motility toward sterile-filtered colonic contents (P=.010), decreased intracellular calcium response to ATP (P=.0075), and their co-culture with IPEC-J2 enhanced epithelial restitution versus monoculture (P=.032). Oppositely, EGC from prebiotic-fed jejunal submucosa showed increased intracellular calcium response (P=.050) and their co-culture with IPEC-J2 did not enhance restitution as efficiently (P=.33) as those from control-fed jejunal submucosa (P=.019). For future study, prebiotic-fed colonic submucosal EGC differentially secreted 13 proteins of interest versus control-fed. Preliminary results indicate dietary oligosaccharides in neonates exert different effects in the jejunum versus the colon on EGC network development and phenotype, and on epithelial restitution in vivo and in vitro. Ongoing work to understand microbiome- EGC-epithelial interactions during postnatal development may lead to novel preventative and clinical practices to improve intestinal health in vulnerable neonates.
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.
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 3 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.
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.
Background: Esophagitis with eosinophilia, inflammation, and fibrosis represents a chronic condition in humans with food allergies. Objective: In this investigation we asked whether esophagitis with an eosinophilic component is observed in young pigs rendered allergic to hen egg white protein (HEWP). Methods: Food allergy was induced in young pigs using two protocols. In one protocol, sensitized pigs were challenged by gavage with a single dose of HEWP. Clinical signs were monitored for 24 h and then gastrointestinal (GI) tissues were collected for histological examination. The phenotype of circulating, ovalbumin (OVA)-specific T cells also was examined in HEWP-challenged animals. In the second protocol, sensitized animals were fed HEWP for 28 days. Animals were then examined by endoscopy and gastrointestinal tissues collected for histological examination. Results: In pigs challenged by gavage with HEWP, clinical signs were noted in 5/6 pigs including diarrhea, emesis, and skin rash. Clinical signs were not seen in any control group. Histological analysis revealed significant levels of esophageal eosinophilic infiltration (p<0.05) in 4/6 of these animals, with two also displaying eosinophilic infiltration in the stomach. Eosinophils were not increased in ileum or colon samples. Increased numbers of circulating, OVA-specific CD4+ T cells also were observed in pigs that received HEWP by gavage. In the group of animals fed HEWP, endoscopy revealed clinical signs of esophagitis including edema, granularity, white spots, and furrowing, while histology revealed edema, immune cell infiltration and basal zone hyperplasia. Conclusions and clinical relevance: Food allergy in the pig can be associated with esophagitis based on histological and endoscopic findings, including eosinophilic infiltration. The young pig may, therefore, be a useful large animal model for the study of eosinophilic esophagitis in humans.