Amanda Ziegler

Breaches in the intestinal barrier lead to sepsis and death if epithelial coverage is not rapidly restored, particularly in neonates. The reason for higher mortality in infants with intestinal injury has not been explained. High infant mortality results from diseases associated with ischemia/ reperfusion (I/R) injury, including necrotizing enterocolitis. Animals also suffer from diseases marked by epithelial sloughing and high mortality, including PED virus in suckling piglets. In juvenile pigs, we have studied rapid intestinal repair by epithelial cell migration (restitution). However, in preliminary studies, we have shown an age-dependent deficiency in the recovery of barrier function following ischemic injury with a near-total lack of restitution in suckling neonates. This is associated with an immature enteric glial cell (EGC) network, which has recently been shown to play a pivotal role in intestinal regeneration. We have also shown positive changes in the microbiome in response to supplementing formula with oligosaccharides. Our central hypothesis is that intestinal repair of ischemic injury is deficient in neonates as a result of a failure of epithelial restitution, which can be rescued by paracrine signaling from maturation of the EGC network in response to a microbiome conditioned by increasing nutritional oligosaccharides. We will test this hypothesis with the following specific aims: 1) Determine age-dependent differences in the EGC network and its paracrine factors in normal and I/R-injured porcine mucosa; 2) Rescue neonatal I/R-injured intestinal mucosa with exogenous application of species-specific EGC soluble factors; 3) Increase EGC numbers and paracrine factors by delivering increased nutritional oligosaccharides to the gut. To examine these specific aims, we will use our unique age-dependent porcine model of mucosal repair, primary culture of porcine EGC, and state of the art imaging techniques for glial cells, including immunolabeling-enabled three-dimensional imaging of solvent-cleared organs (iDISCO). Additionally, we will deliver oligosaccharide-fortified formula with high throughput screening of the microbiome in conjunction with analyses of the EGC network. We expect to identify nutritionally rescuable deficiencies in EGC-directed mechanisms of intestinal recovery.

Our long-term goal is to understand the interplay between nutrition and gut health in order to address major early life intestinal health issues of swine. In preliminary studies, recovery of injured mucosa was impaired in suckling versus weanling pigs, and we have linked this to an underdeveloped component of the enteric nervous system, the enteric glial cell network. This has led us to investigate oligosaccharides, which we have shown alter the microbiome to induce maturation of enteric neural elements involved in gut barrier function. Our central hypothesis is that creep feeding oligosaccharides to suckling pigs will accelerate postnatal microbial colonization, thereby enhancing glial cell maturation and reducing inflammation to promote efficient intestinal barrier maintenance and repair in the peri-weaning period in response to the health challenges of ischemic injury (suckling piglets) or weaning stress (weanling pigs). Our specific objectives are: 1) Determine the ability of creep fed prebiotic oligosaccharides to accelerate postnatal microbial colonization, hasten the development of the EGC network, and enhance intestinal barrier function; 2) Determine if supplementation of creep fed prebiotic oligosaccharides hastens recovery of mucosal barrier function in ischemic-injured suckling intestine by stimulating EGC-regulated restitution; 3) Determine if supplementation of prebiotic oligosaccharides reduces the impact of weaning stress on mast cell infiltration, intestinal health, and growth performance. This work is applicable to the swine industry using a commercial creep feeding system. Our studies will assess intestinal health using state-of-the-art electrophysiologic, cellular, and immunolabeling approaches to define response of the gut to injury and stress.

Postoperative ileus (POI) is a devastating complication of equine colic surgery, and to date, no universally effective treatment exists. It has been theorized that surgical manipulation and luminal distension can decrease intestinal barrier function, allowing bacterial products to enter from the lumen and contribute to the inflammation of the muscularis that is a cornerstone of POI pathophysiology. As enteric glial cells (EGC) are an essential cell population for sensing the intestinal environment and controlling signaling pathways, they provide a potential therapeutic target for the inflammatory component of POI. A pathway of IL-1???????? stimulated EGC releasing IL-6 has previously been described in a rodent model, and IL-6 is capable of altering barrier function. Our preliminary studies demonstrate both an increase in IL-1???????? within mucosa of surgically manipulated porcine intestine and increased EGC IL-1 receptor (IL-1R) expression in small intestine resection margins of horses that develop POI. Therefore, we hypothesize that in equine POI, IL-1???????? stimulated EGC release IL-6 that quickly decreases intestinal barrier function. First, the association between EGC IL-1 signaling and POI will be determined by demonstrating that horses with POI following colic surgery will have increased EGC IL-1R expression and increased levels of IL-1???????? within their small intestinal mucosa. Further, the effect of IL-1???????? stimulated equine EGC on intestinal barrier function in vitro will be evaluated. A novel culture of purified primary equine EGC exposed to IL-1???????? will be used to measure IL-6 secretion. Further, a coculture with equine intestinal epithelial cells will be utilized to observe changes to barrier permeability. This proposed study will provide critical novel insight into the role of EGC IL-1 signaling and barrier permeability in the early pathophysiology of POI, and also highlight a potential biomarker and therapeutic target.

Colic places a massive financial burden on the horse owners of North Carolina each year, and foals with colic are noted to survive less frequently than adult horses. One specific type of colic, when the intestine becomes twisted, can only be corrected with surgery. A large amount of equine veterinary research is focused on improving survival rates for horses that require colic surgery, but to do this, we must better understand what happens at the cellular level during injury and repair. Of particular interest is the inner lining of the intestine, a single layer of cells that form a barrier to prevent the bacteria in the gut from crossing into the blood stream, which can lead to organ failure and death. This single layer of cells (the intestinal barrier) renews in healthy horses about once a week. When the intestine becomes twisted, the blood supply to that area is lost, and these cells die rapidly creating holes in the barrier that allow bacteria into the horse???s body. While surgery restores the blood flow to the area, our research focuses more on helping the intestinal barrier repair itself faster. We think that this repair is controlled, at least in part, by a network of nervous system cells in the intestine called the ???gut brain???. Our lab uses federal funds to study the pig, as a translational model for equine and human health, with a subsequent need to perform equine studies to ensure relevance to horses. We have noticed a difference in barrier repair with regards to age in the pig, and a difference in the gut brain when comparing suckling pigs with weaned pigs. We have adapted the imaging techniques we used in the pig for use in the horse, and have successfully imaged the gut brain in adult equine intestinal tissue. Now, we need assistance in acquiring foal tissue samples, so that we may identify any differences between the gut brain of foals and adults. This is a critical first step in identifying cell types that play a role in repairing the intestinal barrier following colic and colic surgery.

Intestinal diseases which involve ischemia such as volvulus or necrotizing enterocolitis cause mucosal barrier damage and are associated with poor survival in neonatal patients. The cause of higher mortality in neonates as compared to more mature patients has not been fully explained and represents a critical gap in our knowledge. Using a translational pig model of the human infant, I have found that while juvenile animals (6-weeks-old) repair rapidly by villus contraction and epithelial restitution, barrier repair is markedly impaired in neonates (2-weeks-old), characterized by a complete lack of epithelial restitution. Importantly, I found that the restitution defect in neonates can be rescued by the direct application of homogenized mucosal tissue from ischemia-injured small intestine from juvenile pigs. The mechanisms responsible for the age-dependent defect in restitution in neonates and the components of the juvenile mucosal tissues responsible for rescue of restitution are incompletely understood and form the basis of this proposal. Identifying how specific repair signaling mechanisms mature during the postnatal period has the potential to promote innovative discovery efforts leading to effective clinical interventions in neonatal patients with intestinal disease characterized by defective epithelial barrier function. A mixed population of subepithelial cells modulate epithelial cell functions in health and disease by secreting paracrine factors into the mucosal microenvironment. Of these cells, the subepithelial glial network is known to mature postnatally, and has been shown to directly regulate epithelial repair. I examined the glia within porcine jejunum and found an underdeveloped glial network in mucosa of neonates as compared to juveniles. Additionally, I found that epidermal growth factor and intracellular mediators of epithelial migration annexin A2 and focal adhesion kinase are reduced in neonatal mucosa. In order to study glial-epithelial interactions more closely, I will shift my studies to include advanced in vitro techniques. In a preliminary in vitro experiment, I found that media conditioned by glia cultured from juvenile pigs enhanced wound restitution in IPEC-J2 cells, a neonatal-derived intestinal epithelial cell line. These findings suggest glia may play an important role in postnatal development of barrier repair mechanisms. Therefore, I hypothesize that developmental glial factors within the subepithelial microenvironment signal restitution. To examine age-dependent differences in the effects of glia on epithelial repair, I will optimize and study primary enteric glial (aim 1) culture and primary intestinal epithelial culture on cutting-edge biomimetic collagen hydrogel scaffolds (aim 2) in the pig. As a young investigator completing these studies under new mentorship at a new top-ranking medical institution, I will expand my repertoire of skills in experimental design, execution and analysis to include basic science approaches to epithelial restitution mechanisms using advanced in vitro models. These studies will produce data to support new research grants (R03, R01) as I transition to research independence and provide the foundation for a productive career in comparative biomedical research to advance both human and veterinary medicine.

Intestinal diseases which involve ischemia such as volvulus or necrotizing enterocolitis cause mucosal barrier damage and are associated with poor survival in neonatal patients. The cause of higher mortality in neonates as compared to more mature patients has not been fully explained and represents a critical gap in our knowledge. Using a translational pig model of the human infant, I have found that while juvenile animals (6-weeks-old) repair rapidly by villus contraction and epithelial restitution, whereas barrier repair is markedly impaired neonates (2-weeks-old), characterized by a complete lack of epithelial restitution (2-weeks-old). Importantly, I found that the restitution defect in neonates can be rescued by the direct application of homogenized mucosal tissue from ischemiainjured small intestine from juvenile pigs. The reasons for this age-dependent defect in restitution and the components of the juvenile mucosal tissues responsible for rescue remain to be explained and form the basis of this proposal. Due to a growing body of evidence which implicates subepithelial enteric glial cells (EGC) in promoting mucosal barrier repair via paracrine factors such as proEGF, we examined the EGC within porcine tissues. I found an underdeveloped mucosal EGC network in neonates and increased EGC density in juvenile mucosal homogenates. In a preliminary experiment, I found that media conditioned by EGC cultured from juvenile pigs enhanced wound restitution in IPEC-J2 cells, a neonatal-derived intestinal epithelial cell line. Additionally, I found that EGF and intracellular mediators of IEC migration annexin A2 and FAK are reduced in neonatal mucosa. These findings suggest the EGC network may play an important role in postnatal development of barrier repair mechanisms. Therefore, I hypothesize that agedependent soluble factors from cell populations within the mucosal microenvironment,including EGC, signal injured epithelium to restitute following ischemic injury. I will use in vitro IPEC-J2, enteroid monolayer and EGC co-culture models to examine age-dependent differences in EGC distribution and signaling function and rescue age-dependent defects in mechanisms of restitution in wound-adjacent IECs. Identifying how specific repair signaling mechanisms mature during the postnatal period has the potential to stimulate innovative discovery efforts leading to effective clinical interventions in neonatal patients with intestinal disease characterized by defective epithelial barrier function. As a young investigator completing these studies under new mentorship, I will build on my skills in experimental design, execution and analysis to include additional mechanistic approaches to cell signaling research using in vitro models, namely, to master primary EGC culture and cutting-edge enteroid culture techniques. Data from these studies will support research proposals for career development (K01) and subsequent research career (R03) awards as I transition into a faculty position and establish myself as an independent investigator.

Intestinal diseases which involve ischemia such as volvulus or necrotizing enterocolitis cause mucosal barrier damage and are associated with poor survival in neonatal patients. The cause of higher mortality in neonates as compared to more mature patients has not been fully explained and represents a critical gap in our knowledge. Using a translational pig model of the human infant, I have found that while juvenile animals (6-weeks-old) repair rapidly by villus contraction and epithelial restitution, whereas barrier repair is markedly impaired neonates (2-weeks-old), characterized by a complete lack of epithelial restitution (2-weeks-old). Importantly, I found that the restitution defect in neonates can be rescued by the direct application of homogenized mucosal tissue from ischemiainjured small intestine from juvenile pigs. The reasons for this age-dependent defect in restitution and the components of the juvenile mucosal tissues responsible for rescue remain to be explained and form the basis of this proposal. Due to a growing body of evidence which implicates subepithelial enteric glial cells (EGC) in promoting mucosal barrier repair via paracrine factors such as proEGF, we examined the EGC within porcine tissues. I found an underdeveloped mucosal EGC network in neonates and increased EGC density in juvenile mucosal homogenates. In a preliminary experiment, I found that media conditioned by EGC cultured from juvenile pigs enhanced wound restitution in IPEC-J2 cells, a neonatal-derived intestinal epithelial cell line. Additionally, I found that EGF and intracellular mediators of IEC migration annexin A2 and FAK are reduced in neonatal mucosa. These findings suggest the EGC network may play an important role in postnatal development of barrier repair mechanisms. Therefore, I hypothesize that agedependent soluble factors from cell populations within the mucosal microenvironment,including EGC, signal injured epithelium to restitute following ischemic injury. I will use in vitro IPEC-J2, enteroid monolayer and EGC co-culture models to examine age-dependent differences in EGC distribution and signaling function and rescue age-dependent defects in mechanisms of restitution in wound-adjacent IECs. Identifying how specific repair signaling mechanisms mature during the postnatal period has the potential to stimulate innovative discovery efforts leading to effective clinical interventions in neonatal patients with intestinal disease characterized by defective epithelial barrier function. As a young investigator completing these studies under new mentorship, I will build on my skills in experimental design, execution and analysis to include additional mechanistic approaches to cell signaling research using in vitro models, namely, to master primary EGC culture and cutting-edge enteroid culture techniques. Data from these studies will support research proposals for career development (K01) and subsequent research career (R03) awards as I transition into a faculty position and establish myself as an independent investigator.