@article{rose_simon_gomez-martinez_magness_odle_blikslager_ziegler_2025, title={Single-cell transcriptomics predict novel potential regulators of acute epithelial restitution in the ischemia-injured intestine}, url={https://doi.org/10.1152/ajpgi.00194.2024}, DOI={10.1152/ajpgi.00194.2024}, abstractNote={Intestinal ischemic injury damages the epithelial barrier predisposes patients to life-threatening sepsis unless that barrier is rapidly restored. There is an age-dependency of intestinal recovery in that neonates are the most susceptible to succumb to disease of the intestinal barrier versus older patients. We have developed a pig model that demonstrates age-dependent failure of intestinal barrier restitution in neonatal pigs which can be rescued by the direct application of juvenile pig mucosal tissue, but the mechanisms of rescue remain undefined. We hypothesized that by identifying a subpopulation of restituting enterocytes by their expression of cell migration transcriptional pathways, we can then predict novel upstream regulators of age-dependent restitution response programs. Superficial mucosal epithelial cells from recovering ischemic jejunum of juvenile pigs underwent single cell transcriptomics and predicted upstream regulator CSF-1 was interrogated in our model. A subcluster of absorptive enterocytes expressed several cell migration pathways key to restitution. Differentially expressed genes in this subcluster predicted their upstream regulation by colony stimulating factor-1 (CSF-1). We validated age-dependent induction of CSF-1 by ischemia and documented that CSF-1 and CSF1R co-localized in ischemic juvenile, but not neonatal, wound-adjacent epithelial cells and in the restituted epithelium of juveniles and rescued neonates. Further, the CSF-1 blockade reduced restitution in vitro, and CSF-1 improved barrier function in injured neonatal pig in preliminary ex vivo experiments. These studies validate an approach to inform potential novel therapeutic targets, such as CSF-1, to improve outcomes in neonates with intestinal injury in a unique pig model.}, journal={American Journal of Physiology-Gastrointestinal and Liver Physiology}, author={Rose, Elizabeth C. and Simon, Jeremy M. and Gomez-Martinez, Ismael and Magness, Scott T. and Odle, Jack and Blikslager, Anthony T. and Ziegler, Amanda L.}, year={2025}, month={Mar} }
 @article{awan_bartlett_blakeley-ruiz_richie_ziegler_kleiner_2025, title={Source of dietary protein alters the abundance of proteases, intestinal epithelial and immune proteins both directly and via interactions with the gut microbiota}, url={https://doi.org/10.1101/2025.01.09.632171}, DOI={10.1101/2025.01.09.632171}, abstractNote={Dietary protein has been shown to impact long-term health outcomes differentially depending on its amount and source. It has been suggested that interactions of the gut microbiota with dietary proteins mediate some of the effects of dietary protein on health outcomes. However, it remains unclear what specific host responses drive the health effects of dietary proteins from different plant and animal sources. Additionally, which specific host responses are mediated by interactions of dietary protein source with the gut microbiota and which host responses are caused by dietary proteins directly is not well understood. We used metaproteomics to quantify dietary, host, and microbial proteins in fecal samples of conventional and germ-free mice fed purified dietary protein from six different plant and animal sources, including casein, egg-white, soy, brown rice, pea, and yeast. We characterized differences in the host fecal proteome across the six dietary protein sources as well as between the conventional and germ-free mice for each source to determine how the host responds to the different dietary protein sources and the role of the gut microbiota in mediating these responses. We found that both the source of dietary protein and the presence or absence of the gut microbiota drive the host response to dietary protein source in the fecal host proteome. Host proteins pertaining to immune response, digestion, and barrier function were differentially abundant in different protein sources with and without the gut microbiota. These changes in the host response correlated with changes in microbial composition and differences in protein digestibility. Our results show how dietary protein sources, through their interactions with the gut microbiota, impact several aspects of host physiology.}, author={Awan, Ayesha and Bartlett, Alexandria and Blakeley-Ruiz, J. Alfredo and Richie, Tanner and Ziegler, Amanda and Kleiner, Manuel}, year={2025}, month={Jan} }
 @article{ziegler_caldwell_craig_hellstrom_sheridan_touvron_pridgen_magness_odle_van landeghem_et al._2024, title={Enteric glial cell network function is required for epithelial barrier restitution following intestinal ischemic injury in the early postnatal period}, volume={326}, ISSN={["1522-1547"]}, url={https://doi.org/10.1152/ajpgi.00216.2022}, DOI={10.1152/ajpgi.00216.2022}, abstractNote={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.}, number={3}, journal={AMERICAN JOURNAL OF PHYSIOLOGY-GASTROINTESTINAL AND LIVER PHYSIOLOGY}, author={Ziegler, Amanda L. and Caldwell, Madison L. and Craig, Sara E. and Hellstrom, Emily A. and Sheridan, Anastasia E. and Touvron, Melissa S. and Pridgen, Tiffany A. and Magness, Scott T. and Odle, Jack and Van Landeghem, Laurianne and et al.}, year={2024}, month={Mar}, pages={G228–G246} }
 @article{hellstrom_mckinney-aguirre_gonzalez_ziegler_blikslager_2024, title={Equine Enteric Glial Culture and Application to the Study of A Neural Inflammatory Mechanism in Equine Colic}, volume={10}, ISSN={["1940-087X"]}, DOI={10.3791/67244}, abstractNote={Inflammatory postoperative conditions of equine colic (acute abdomen) contribute not only to increased client cost, patient discomfort, and hospitalization time, but in many cases, prove to be life-threatening. A unique population of intestinal cells, enteric glia, are increasingly acknowledged for their roles in sensing the gastrointestinal environment and communicating with surrounding cell types. Interactions between enteric glia and intestinal epithelia may prove critical in establishing how equine enteric glia can alter the mucosal barrier to modulate inflammation in health and colic. To study this interaction, we present a method of establishing primary equine enteric glial cultures from equine jejunum and exposing the cultures to inflammatory conditions known to be present in colic. Primary enteric glial cultures were obtained from adult horses euthanized for reasons unrelated to colic. Intestinal villi and lamina propria were micro-dissected to expose the submucosa. The isolated submucosa underwent enzymatic digestion with collagenase, protease, and bovine serum albumin for 2-3 h. Next, mechanical digestion involving centrifugation, pipetting, and cell strainers (40-100 µm), yielded a pellet used for plating on 0.05 mg/mL poly-L-lysine-coated wells at a concentration of ~400,000 cells/300 µL of media. Following confluence and first passage, the enteric glial cells were then exposed to equine recombinant IL-1β (0, 10, 25 ng) for 24 h. To model epithelial-glial interactions at the time of colic, medium conditioned by either control or treated enteric glia was added directly to confluent equine jejunal monolayers while measuring transepithelial electrical resistance (TEER) using a dual-electrode EndOhm chamber. These data demonstrate just one of many potential impactful applications of equine enteric glial culture.}, number={212}, journal={JOVE-JOURNAL OF VISUALIZED EXPERIMENTS}, author={Hellstrom, Emily and McKinney-Aguirre, Caroline and Gonzalez, Liara and Ziegler, Amanda and Blikslager, Anthony}, year={2024}, month={Oct} }
 @article{boston_wang_lin_kim_fellner_scott_ziegler_van landeghem_blikslager_odle_2024, title={Prebiotic galactooligosaccharide improves piglet growth performance and intestinal health associated with alterations of the hindgut microbiota during the peri-weaning period}, volume={15}, ISSN={["2049-1891"]}, url={https://doi.org/10.1186/s40104-024-01047-y}, DOI={10.1186/s40104-024-01047-y}, abstractNote={Abstract Background Weaning stress reduces growth performance and health of young pigs due in part to an abrupt change in diets from highly digestible milk to fibrous plant-based feedstuffs. This study investigated whether dietary galactooligosaccharide (GOS), supplemented both pre- and post-weaning, could improve growth performance and intestinal health via alterations in the hindgut microbial community. Methods Using a 3 × 2 factorial design, during farrowing 288 piglets from 24 litters received either no creep feed (FC), creep without GOS (FG–) or creep with 5% GOS (FG+) followed by a phase 1 nursery diet without (NG–) or with 3.8% GOS (NG+). Pigs were sampled pre- (D22) and post-weaning (D31) to assess intestinal measures. Results Creep fed pigs grew 19% faster than controls ( P < 0.01) prior to weaning, and by the end of the nursery phase (D58), pigs fed GOS pre-farrowing (FG+) were 1.85 kg heavier than controls ( P < 0.05). Furthermore, pigs fed GOS in phase 1 of the nursery grew 34% faster ( P < 0.04), with greater feed intake and efficiency. Cecal microbial communities clustered distinctly in pre- vs. post-weaned pigs, based on principal coordinate analysis ( P < 0.01). No effects of GOS were detected pre-weaning, but gruel creep feeding increased Chao1 α-diversity and altered several genera in the cecal microbiota ( P < 0.05). Post-weaning, GOS supplementation increased some genera such as Fusicatenibacter and Collinsella, whereas others decreased such as Campylobacter and Frisingicoccus ( P < 0.05). Changes were accompanied by higher molar proportions of butyrate in the cecum of GOS-fed pigs ( P < 0.05). Conclusions Gruel creep feeding effectively improves suckling pig growth regardless of GOS treatment. When supplemented post-weaning, prebiotic GOS improves piglet growth performance associated with changes in hindgut microbial composition.}, number={1}, journal={JOURNAL OF ANIMAL SCIENCE AND BIOTECHNOLOGY}, author={Boston, Timothy E. and Wang, Feng and Lin, Xi and Kim, Sung Woo and Fellner, Vivek and Scott, Mark F. and Ziegler, Amanda L. and Van Landeghem, Laurianne and Blikslager, Anthony T. and Odle, Jack}, year={2024}, month={Jun} }
 @article{caldwell_cook_mariant_touvron_odle_blikslager_ziegler_van landeghem_2024, title={Protocol Protocol to culture enteric glial cells from the submucosal and myenteric plexi of neonatal and juvenile pig colons}, volume={5}, ISSN={["2666-1667"]}, DOI={10.1016j.xpro.2024.103057}, number={2}, journal={STAR PROTOCOLS}, author={Caldwell, Madison L. and Cook, Caleb A. and Mariant, Chloe L. and Touvron, Melissa and Odle, Jack and Blikslager, Anthony T. and Ziegler, Amanda L. and Van Landeghem, Laurianne}, year={2024}, month={Jun} }
 @misc{elane_bauck_hobbs_king_fields_ziegler_blikslager_2024, title={Review of venipuncture and intravenous catheterization techniques in pigs}, volume={262}, ISSN={["1943-569X"]}, url={http://dx.doi.org/10.2460/javma.24.03.0169}, DOI={10.2460/javma.24.03.0169}, abstractNote={Abstract The number of companion pigs in the US is increasing, as is the frequency with which they present to primary companion care practices. However, pigs are often an understudied species in veterinary curricula, and many students graduate from veterinary school with minimal porcine handling experience. Coupled with the poor peripheral vascular access associated with pigs, this presents a challenge for many new graduates and other primary care veterinarians seeking to improve their knowledge of porcine handling, anatomy, and medical care. Furthermore, much of the available veterinary literature regarding porcine venous access is dated, limited to technical notes, or inaccessible to many primary care veterinarians. This review aims to supplement this lack of knowledge by discussing techniques in restraint, sedation, venipuncture, and catheterization of companion pigs as a reference for veterinarians and researchers alike.}, number={10}, journal={JAVMA-JOURNAL OF THE AMERICAN VETERINARY MEDICAL ASSOCIATION}, author={Elane, George L. and Bauck, Anje G. and Hobbs, Kallie J. and King, Ailbhe and Fields, Claire and Ziegler, Amanda and Blikslager, Anthony}, year={2024}, month={Oct}, pages={1388–1396} }
 @article{rose_simon_gomez-martinez_magness_odle_blikslager_ziegler_2024, title={Single-cell transcriptomics predict novel potential regulators of acute epithelial restitution in the ischemia-injured intestine}, url={https://doi.org/10.1101/2024.06.28.601271}, DOI={10.1101/2024.06.28.601271}, abstractNote={ABSTRACT Intestinal ischemic injury damages the epithelial barrier predisposes patients to life-threatening sepsis unless that barrier is rapidly restored. There is an age-dependency of intestinal recovery in that neonates are the most susceptible to succumb to disease of the intestinal barrier versus older patients. We have developed a pig model that demonstrates age-dependent failure of intestinal barrier restitution in neonatal pigs which can be rescued by the direct application of juvenile pig mucosal tissue, but the mechanisms of rescue remain undefined. We hypothesized that by identifying a subpopulation of restituting enterocytes by their expression of cell migration transcriptional pathways, we can then predict novel upstream regulators of age-dependent restitution response programs. Superficial mucosal epithelial cells from recovering ischemic jejunum of juvenile pigs were processed for single cell RNA sequencing analysis, and predicted upstream regulators were assessed in a porcine intestinal epithelial cell line (IPEC-J2) and banked tissues. A subcluster of absorptive enterocytes expressed several cell migration pathways key to restitution. Differentially expressed genes in this subcluster predicted their upstream regulation included colony stimulating factor-1 (CSF-1). We validated age-dependent induction of CSF-1 by ischemia and documented that CSF-1 and CSF1R co-localized in ischemic juvenile, but not neonatal, wound-adjacent epithelial cells and in the restituted epithelium of juveniles and rescued (but not control) neonates. Further, the CSF1R inhibitor BLZ945 reduced restitution in scratch wounded IPEC-J2 cells. These studies validate an approach to inform potential novel therapeutic targets, such as CSF-1, to improve outcomes in neonates with intestinal injury in a unique pig model. NEW & NOTEWORTHY These studies validate an approach to identify and predict upstream regulation of restituting epithelium in a unique pig intestinal ischemic injury model. Identification of potential molecular mediators of restitution, such as CSF-1, will inform the development of targeted therapeutic interventions for medical management of patients with ischemia-mediated intestinal injury.}, author={Rose, Elizabeth C. and Simon, Jeremy M. and Gomez-Martinez, Ismael and Magness, Scott T. and Odle, Jack and Blikslager, Anthony T. and Ziegler, Amanda L.}, year={2024}, month={Jun} }
 @article{reeder_zarlenga_ziegler_dyer_2024, title={Transcriptional responses consistent with perturbation in dermo-epidermal homeostasis in septic sole ulceration}, volume={107}, ISSN={["1525-3198"]}, url={https://doi.org/10.3168/jds.2023-24578}, DOI={10.3168/jds.2023-24578}, abstractNote={The aim of this study was to evaluate transcriptional changes in the sole epidermis and dermis of bovine claws with septic sole ulceration of the lateral claw. Assessment included changes in transcripts orchestrating epidermal homeostatic processes, including epidermal proliferation, differentiation, inflammation, and cell signaling. Sole epidermis and dermis samples were removed from region 4 of lesion-bearing lateral and lesion-free medial claws of pelvic limbs in multiparous, lactating Holstein cows. Control sole epidermis and dermis samples were obtained from region 4 of lateral claws of normal pelvic limbs. Transcript abundances were evaluated by real-time PCR, and relative expression analyzed by ANOVA. Relative to normal lateral claws, sole epidermis and dermis in ulcer-bearing claws exhibited downregulation of genes associated with growth factors, growth factor receptors, activator protein 1 (AP-1) and proto-oncogene (CMYC) transcription components, cell cycle elements, lateral cell-to-cell signaling elements, and structures of early and late keratinocyte differentiation. These changes were accompanied by upregulation of proinflammatory transcripts interleukin 1 α (IL1A), interleukin1 β (IL1B), interleukin 1 receptor 1 (IL1R1), inducible nitric oxide synthase (NOS2), the inflammasome components NOD-like receptor protein 3 (NLRP3), pyrin and caspase recruitment domain (PYCARD), caspase-1 interleukin converting enzyme (CASPASE), the matrix metalloproteinases (MMP2 and MMP9), and the anti-inflammatory genes interleukin 1 receptor antagonist (IL1RN) and interleukin1 receptor 2 (IL1R2). Transcript abundance varied across epidermis and dermis from the ulcer center, margin, and epidermis and dermis adjacent to the lesion. Sole epidermis and dermis of lesion-free medial claws exhibited changes paralleling those in the adjacent lateral claws in an environment lacking inflammatory transcripts and downregulated IL1A, interleukin 18 (IL18), tumor necrosis factor α (TNFA), and NOS2. These data imply perturbations in signal pathways driving epidermal proliferation and differentiation are associated with, but not inevitably linked to epidermis and dermis inflammation. Further work is warranted to better define the role of crushing tissue injury, sepsis, metalloproteinase activity, and inflammation in sole ulceration.}, number={10}, journal={JOURNAL OF DAIRY SCIENCE}, author={Reeder, T. L. and Zarlenga, D. S. and Ziegler, A. L. and Dyer, R. M.}, year={2024}, month={Oct}, pages={8432–8451} }
 @article{boston_wang_xi_kim_fellner_scott_ziegler_van landeghem_blikslager_odle_2023, title={Effects of Prebiotic Galactooligosaccharide (GOS) on Hindgut Microbial Composition of Pigs During the Peri-Weaning Period}, volume={101}, ISSN={["1525-3163"]}, DOI={10.1093/jas/skad281.228}, abstractNote={Abstract Galactooligosaccharide (GOS) is a prebiotic containing beta-linked galactose oligomers of 2 to 8 units. Previously, GOS positively impacted post-weaning growth performance and altered jejunal morphology. In this study, GOS-enriched whey permeate (Milk Specialties Global, Eden Prairie MN) was supplemented to piglets in farrowing and nursery phases. To maximize pre-weaning GOS intake, novel gruel creep feeders were used. Piglets from 24 litters received either no creep feed (NC), creep without GOS (CG-) or creep with 5.0% GOS (CG+) followed by a phase 1 nursery diet without (NG-) or with 3.8% GOS (NG+). At d 22 (pre-weaning) and d 31 (post-weaning), 6 pigs per treatment were euthanized for sample collection. DNA was extracted from cecal and rectal swabs for downstream 16S rRNA sequencing via Illumina MiSeq (Zymo Research Corp, Irvine CA). An amplicon sequence variant (ASV) table was created using the DADA2 pipeline in R and taxonomy assignment was performed using the SILVA database. Average sequencing depth after filtering was 138,323 and low sequencing counts were discarded based on 20% prevalence. Data were analyzed using the lmer and adonis procedures in RStudio ver. 4.2.2. Analysis of 16S rRNA genes did not detect pre- or post-weaning GOS effects on Chao1, Simpson, or Shannon alpha diversity measures (P > 0.1). Similarly, no pre-weaning GOS effects were detected in Principal Coordinates Analysis (PCoA) beta diversity plots (P > 0.1); however, pigs fed GOS post-weaning segregated from controls (P < 0.01). The relative abundance of several genera including Fusicatenbacter and Collinsella increased by 2.0 and 1.3-fold, respectively, in the cecum of NG+ pigs, whereas, Bacteroides decreased by 3.8-fold (FDR, P < 0.05). In the rectum, relative abundance of Corprococcus decreased by 2.0-fold and Fusicatenbacter increased by 1.5-fold in NG+ pigs compared with NG- pigs (FDR, P < 0.05). Collectively, these genera in the cecum and rectum comprise of less than 3% of the total community. In contrast, lactobacillus in the cecum (33%) and rectum (21%) had a large relative abundance but was unaffected by treatment. We conclude that supplementation of GOS in the post-weaning phase 1 nursery diet alters the hindgut microbial community and may contribute to improvements in growth performance.}, journal={JOURNAL OF ANIMAL SCIENCE}, author={Boston, Timothy E. and Wang, Feng and Xi, Lin and Kim, Sung Woo and Fellner, Vivek and Scott, Mark F. and Ziegler, Amanda L. and Van Landeghem, Laurianne and Blikslager, Anthony T. and Odle, Jack}, year={2023}, month={Nov}, pages={187–188} }
 @article{boston_wang_xi_kim_fellner_scott_ziegler_van landeghem_blikslager_odle_2023, title={Effects of Prebiotic Galactooligosaccharide (Gos) on Piglet Growth and Jejunal Morphology During the Peri-Weaning Period}, volume={101}, ISSN={["1525-3163"]}, DOI={10.1093/jas/skad341.313}, abstractNote={Abstract Galactooligosaccharide (GOS) is a specific prebiotic that is enzymatically synthesized from lactose to form beta-linked oligosaccharides containing 2-8 galactose units. In this study, GOS-enriched whey permeate (Milk Specialties Global, Eden Prairie MN) was supplemented to piglets in lactation and nursery phases. To maximize pre-weaning GOS intake, novel gruel creep feeders were utilized. Using a 3x2 factorial design, piglets from 24 litters received either no creep feed (NC), creep without GOS (CG-) or creep with 5% GOS (CG+) followed by a phase 1 nursery diet without (NG-) or with 3.8% GOS (NG+). After 1 week, pigs were fed common phase 2 and phase 3 diets. At d 22 (pre-weaning) and d 31 (post-weaning), 6 pigs per treatment were euthanized for intestinal measurements. Pre-weaning, creep fed pigs grew 19% faster than controls (P < 0.01) but GOS effects were not detected (P > 0.1). In contrast, pigs fed GOS in phase 1 grew 34 % faster than controls (P < 0.04), irrespective of creep treatment (interaction P > 0.1), and with corresponding greater intakes (P < 0.06). These GOS effects were sustained for overall nursery performance. Furthermore, overall ADG of CG+ piglets in the nursery tended to be greatest (P = 0.09), gaining 361g/d, followed by NC (324 g/d) and CG- (310 g/d) treatments (Table 1). No effects on jejunal morphology were detected at d 22, although there was an effect of age with decreased villi length, villus area, villi:crypt ratio and increased crypt depth at d 31 (P < 0.01). Supplementation of GOS in phase 1 increased villus length (36%) and area (51%) but only in pigs previously fed the control creep (CG-) diet (interaction, P < 0.01). Treatment effects on cecal pH and VFA concentrations were not detected, although there was an effect of age with a decrease in pH (P < 0.01) and increase in propionate and butyrate concentrations post-weaning (P < 0.01). We conclude that gruel creep feeding increases weight gain regardless of GOS treatment and that nursery growth and intestinal morphology are improved by post-weaning GOS supplementation. Funded in part by Milk Specialties Global, USDA-NIFA 2022-67015-37125 and Hatch 1016618.}, journal={JOURNAL OF ANIMAL SCIENCE}, author={Boston, Timothy E. and Wang, Feng and Xi, Lin and Kim, Sung Woo and Fellner, Vivek and Scott, Mark F. and Ziegler, Amanda L. and Van Landeghem, Laurianne and Blikslager, Anthony T. and Odle, Jack}, year={2023}, month={Oct}, pages={276–277} }
 @article{rivera_bilton_burclaff_czerwinski_liu_trueblood_hinesley_breau_deal_joshi_et al._2023, title={Hypoxia Primes Human ISCs for Interleukin-Dependent Rescue of Stem Cell Activity}, volume={16}, ISSN={["2352-345X"]}, DOI={10.1016/j.jcmgh.2023.07.012}, abstractNote={Background and AimsHypoxia 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).MethodshISCs 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.ResultsThe 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 IL1β, IL2, IL4, IL6, IL10, IL13, and IL25 and rescued hISC activity caused by 24 hours of hypoxia.ConclusionsHypoxia 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. 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). 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. 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 IL1β, IL2, IL4, IL6, IL10, IL13, and IL25 and rescued hISC activity caused by 24 hours of hypoxia. 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.}, number={5}, journal={CELLULAR AND MOLECULAR GASTROENTEROLOGY AND HEPATOLOGY}, author={Rivera, Kristina R. and Bilton, R. Jarrett and Burclaff, Joseph and Czerwinski, Michael J. and Liu, Jintong and Trueblood, Jessica M. and Hinesley, Caroline M. and Breau, Keith A. and Deal, Halston E. and Joshi, Shlok and et al.}, year={2023}, pages={823–846} }
 @article{ziegler_erwin_caldwell_touvron_pridgen_magness_odle_landeghem_blikslager_2022, title={Enteric Glial Cell Network Function is Required for Epithelial Barrier Restitution following Intestinal Ischemic Injury in the Early Postnatal Period}, url={https://doi.org/10.1101/2022.11.04.514575}, DOI={10.1101/2022.11.04.514575}, abstractNote={ABSTRACT 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 assessed ex vivo in Ussing chamber using the glia-specific inhibitor fluoroacetate, and in vivo by 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 & 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 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.}, author={Ziegler, Amanda L. and Erwin, Sara and Caldwell, Madison L. and Touvron, Melissa S. and Pridgen, Tiffany A. and Magness, Scott T. and Odle, Jack and Landeghem, Laurianne Van and Blikslager, Anthony T.}, year={2022}, month={Nov} }
 @article{boger_sheridan_ziegler_blikslager_2022, title={Mechanisms and modeling of wound repair in the intestinal epithelium}, volume={6}, ISSN={["2168-8370"]}, url={https://doi.org/10.1080/21688370.2022.2087454}, DOI={10.1080/21688370.2022.2087454}, abstractNote={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.}, journal={TISSUE BARRIERS}, author={Boger, Kasey D. and Sheridan, Ana E. and Ziegler, Amanda L. and Blikslager, Anthony T.}, year={2022}, month={Jun} }
 @article{erwin_clark_dechant_aitken_hassel_blikslager_ziegler_2022, title={Multi-Institutional Retrospective Case-Control Study Evaluating Clinical Outcomes of Foals with Small Intestinal Strangulating Obstruction: 2000-2020}, volume={12}, ISSN={["2076-2615"]}, url={https://doi.org/10.3390/ani12111374}, DOI={10.3390/ani12111374}, abstractNote={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.}, number={11}, journal={ANIMALS}, publisher={MDPI AG}, author={Erwin, Sara J. and Clark, Marley E. and Dechant, Julie E. and Aitken, Maia R. and Hassel, Diana M. and Blikslager, Anthony T. and Ziegler, Amanda L.}, year={2022}, month={Jun} }
 @misc{rose_blikslager_ziegler_2022, title={Porcine Models of the Intestinal Microbiota: The Translational Key to Understanding How Gut Commensals Contribute to Gastrointestinal Disease}, volume={9}, ISSN={["2297-1769"]}, DOI={10.3389/fvets.2022.834598}, abstractNote={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.}, journal={FRONTIERS IN VETERINARY SCIENCE}, author={Rose, Elizabeth C. and Blikslager, Anthony T. and Ziegler, Amanda L.}, year={2022}, month={Mar} }
 @article{erwin_blikslager_ziegler_2021, title={Age-Dependent Intestinal Repair: Implications for Foals with Severe Colic}, volume={11}, ISSN={["2076-2615"]}, url={https://doi.org/10.3390/ani11123337}, DOI={10.3390/ani11123337}, abstractNote={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.}, number={12}, journal={ANIMALS}, author={Erwin, Sara J. and Blikslager, Anthony T. and Ziegler, Amanda L.}, year={2021}, month={Dec} }
 @misc{hellstrom_ziegler_blikslager_2021, title={Postoperative Ileus: Comparative Pathophysiology and Future Therapies}, volume={8}, ISSN={["2297-1769"]}, DOI={10.3389/fvets.2021.714800}, abstractNote={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.}, journal={FRONTIERS IN VETERINARY SCIENCE}, author={Hellstrom, Emily A. and Ziegler, Amanda L. and Blikslager, Anthony T.}, year={2021}, month={Sep} }
 @article{rose_odle_blikslager_ziegler_2021, title={Probiotics, Prebiotics and Epithelial Tight Junctions: A Promising Approach to Modulate Intestinal Barrier Function}, volume={22}, ISSN={1422-0067}, url={http://dx.doi.org/10.3390/ijms22136729}, DOI={10.3390/ijms22136729}, abstractNote={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.}, number={13}, journal={International Journal of Molecular Sciences}, publisher={MDPI AG}, author={Rose, Elizabeth C. and Odle, Jack and Blikslager, Anthony T. and Ziegler, Amanda L.}, year={2021}, month={Jun}, pages={6729} }
 @article{sheridan_pridgen_odle_van landeghem_blikslager_ziegler_2020, title={A Glial Cell Inhibitor Blocks Epithelial Barrier Repair in a Pig Model of Intestinal Ischemia}, volume={34}, ISSN={["1530-6860"]}, DOI={10.1096/fasebj.2020.34.s1.02030}, abstractNote={The gut epithelium functions as a barrier against toxic luminal contents which can repair efficiently after injury to prevent systemic illness and death. However, we have shown that repair is severely hindered in neonates as compared to juveniles in our translational pig model of intestinal ischemia. The subepithelial enteric glial cell (EGC) network is known to promote epithelial repair by paracrine signaling mechanisms. This EGC network develops postnatally; therefore, we believe this repair defect in neonates is due to an underdeveloped EGC network. In support of this, we hypothesize that EGC inhibition in juveniles will block epithelial barrier repair after ischemic injury mimicking the neonatal phenotype. Ischemia‐injured jejunum of juvenile pigs was recovered ex vivo with and without fluoroacetate (FA), a glial inhibitor. Transepithelial electrical resistance (TEER) was monitored as a measure of barrier function, and tissues were collected for imaging analysis and primary EGC culture. FA inhibited TEER recovery at all tested concentrations (P<0.0001). Histology showed 500μM FA optimally inhibits epithelial repair (P=0.0196) without directly damaging the epithelium (P=0.5509). Cellular metabolism marker c‐fos has been optimized for immunofluorescence in control tissues and ongoing imaging work will validate selective inhibition of EGC metabolism by 500μM FA. In addition, pig EGC isolation and culture has been optimized, so that future work will confirm inhibitory effects of FA on EGC signaling functions in vitro . Understanding the development of glial‐epithelial crosstalk in barrier repair will ultimately guide novel clinical solutions to improve outcomes in neonatal patients affected by intestinal injury. Support or Funding Information UNC CGIBD Large Animal Models Core (P30 DK034987), UNC CGIBD Basic Science Research Training Fellowship (NIH T32 5T32DK007737), UNC CGIBD Pilot Feasibility Grant (P30 DK034987), USDA National Institute of Food and Agriculture (Projects 1007263 and 07985)}, number={S1}, journal={FASEB JOURNAL}, publisher={Wiley}, author={Sheridan, Ana and Pridgen, Tiffany and Odle, Jack and Van Landeghem, Laurianne and Blikslager, Anthony and Ziegler, Amanda}, year={2020}, month={Apr} }
 @inproceedings{sheridan_pridgen_odle_landeghem_blikslager_ziegler_2020, place={San Diego, CA, USA}, title={A Glial Cell Inhibitor Blocks Epithelial Barrier Repair in a Pig Model of Intestinal Ischemia}, booktitle={Experimental Biology}, author={Sheridan, A.E. and Pridgen, T.A. and Odle, J. and Landeghem, L.Van and Blikslager, A.T. and Ziegler, A.L.}, year={2020}, month={Apr} }
 @inproceedings{ziegler_sheridan_pridgen_odle_landeghem_blikslager_2020, place={Washington, DC, USA}, title={An age-dependent, rescuable defect in intestinal barrier repair is associated with an immature enteric glial network in a neonatal pig model of intestinal ischemia}, booktitle={Translational Science 2020}, author={Ziegler, A.L. and Sheridan, A.E. and Pridgen, T.A. and Odle, J. and Landeghem, LVan and Blikslager, A.T.}, year={2020}, month={Apr} }
 @article{ziegler_blikslager_2020, title={Effects of Environmental Acclimation versus Transport Stress on Barrier Recovery in a Pig Model of Intestinal Ischemia and Repair}, volume={34}, ISSN={["1530-6860"]}, DOI={10.1096/fasebj.2020.34.s1.09380}, abstractNote={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 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 for 30‐minutes in 8–10‐week‐old pigs which had been transported and allowed to acclimate to a biomedical research housing environment for 3‐days prior to injury or been transported immediately prior to injury. Jejunal mucosa was then allowed to recover ex vivo in Ussing chambers while transepithelial electrical resistance (TEER) and mannitol flux were measured, and epithelial integrity was assessed by histomorphometry. In uninjured mucosa, there was no difference in basal TEER (P=0.8801) or epithelial integrity on histology between groups (P=0.5686), however, acclimated pigs had increased flux as compared to transported pigs during the first hour of ex vivo incubation (P=0.0186). Ischemia‐injured mucosa of acclimated pigs had less robust TEER recovery ex vivo (P=0.0101) as compared to transported pigs, but an increased initial flux in this group was significantly reduced during recovery (P=0.0025). Ischemia induced greater epithelial loss in transported pigs as compared to acclimated pigs (68.5% versus 87.8% epithelial coverage, P<0.0001), yet both groups restored control levels of epithelial coverage after 120‐minutes ex vivo recovery. These results indicate that acute transport stress on the day of experimental intestinal injury modeling may increase mucosal susceptibility to epithelial loss, but also prime the tissue for a more robust barrier repair response. Brief environmental acclimation, on the other hand, appears to increase intestinal permeability to molecular flux in the absence of injury, while possibly having a protective effect on epithelial loss during injury. These are important considerations for appropriate study design when utilizing highly translational pig models for intestinal barrier research. Support or Funding Information NIH K01 OD 028207, NIH P30 DK 034987, NIH‐NICHD R01 HD095876, USDA‐NIFA VMCG‐0065, USDA‐NIFA 1007263.}, journal={FASEB JOURNAL}, author={Ziegler, Amanda and Blikslager, Anthony}, year={2020}, month={Apr} }
 @inproceedings{ziegler_blikslager_2020, place={San Diego, CA, USA}, title={Effects of Environmental Acclimation versus Transport Stress on Barrier Recovery in a Pig Model of Intestinal Ischemia and Repair}, booktitle={Experimental Biology}, author={Ziegler, A.L. and Blikslager, A.T.}, year={2020}, month={Apr} }
 @article{shapiro_ziegler_odle_van landeghem_blikslager_2020, title={Effects of Oligosaccharide Supplementation on Intestinal Morphology and Enteric Glial Cell Marker Expression in a Neonatal Pig Model}, volume={34}, ISSN={["1530-6860"]}, DOI={10.1096/fasebj.2020.34.s1.09457}, abstractNote={We have shown that oligosaccharide supplementation can modulate the gut flora in neonates, and the microbiome is known to drive the maturation of the enteric glial cell (EGC) network postnatally. Here, we test the effects of dietary oligosaccharides on postnatal changes in the EGC network and intestinal morphology in a neonatal pig model.After suckling colostrum for 24‐hours, one‐day‐old pigs were grouped onto one of three formula‐based diets: control, high oligosaccharide (1:1 mixture of galactooligosaccharide and polydextrose, 8g/L), or low oligosaccharide (4g/L). Small intestine and colon samples were collected at 1‐ 7‐ 14‐ and 21‐ days‐of‐age for western blot and histological analysis. Preliminary histological results indicate a trend toward a decreased small intestinal villus length in the high oligosaccharide group at 7‐days‐of‐age, indicative of accelerated intestinal maturity. Expression of the EGC marker glial fibrillary acidic protein is increased in the small intestinal mucosa at 7‐ and 14‐days‐of‐age in the high oligosaccharide group based on a preliminary western blot (n=1). Following ongoing work to assess crypt morphology and quantify additional EGC markers S100B, PLP‐1, Sox10 in the small intestine and colon, we expect to find increased mucosal expression of EGC markers earlier in postnatal development in the high oligosaccharide group, as well as histological changes consistent with enhanced rates of gut maturation in pigs fed a high oligosaccharide diet. Understanding how dietary inputs drive intestinal development postnatally may improve practices for managing optimal gut heath early in life. Support or Funding Information NIH K01 OD 028207, NIH P30 DK 034987, NIH‐NICHD R01 HD095876, USDA‐NIFA VMCG‐0065}, number={S1}, journal={FASEB JOURNAL}, publisher={Wiley}, author={Shapiro, Lindsey and Ziegler, Amanda and Odle, Jack and Van Landeghem, Laurianne and Blikslager, Anthony}, year={2020}, month={Apr} }
 @inproceedings{shapiro_ziegler_odle_landeghem_blikslager_2020, place={San Diego, CA, USA}, title={Effects of Oligosaccharide Supplementation on Intestinal Morphology and Enteric Glial Cell Marker Expression in a Neonatal Pig Model. }, booktitle={Experimental Biology}, author={Shapiro, L. and Ziegler, A.L. and Odle, J. and Landeghem, L.Van and Blikslager, A.T.}, year={2020}, month={Apr} }
 @article{ziegler_pridgen_blikslager_2020, title={Environmental stressors affect intestinal permeability and repair responses in a pig intestinal ischemia model}, volume={8}, ISSN={["2168-8370"]}, url={https://doi.org/10.1080/21688370.2020.1832421}, DOI={10.1080/21688370.2020.1832421}, abstractNote={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.}, number={4}, journal={TISSUE BARRIERS}, publisher={Informa UK Limited}, author={Ziegler, Amanda L. and Pridgen, Tiffany A. and Blikslager, Anthony T.}, year={2020}, month={Oct} }
 @inproceedings{erwin_dechant_aitken_hassel_ziegler_blikslager_2020, title={Multi-institutional retrospective case-control study evaluating clinical outcomes of foals with small intestinal strangulating obstruction: 2000-2019. }, booktitle={2020 American College of Veterinary Surgeons Surgery E-Summit}, publisher={American College of Veterinary Surgeons Surgery E-Summit}, author={Erwin, S.J. and Dechant, J.E. and Aitken, M.R. and Hassel, D.M. and Ziegler, A.L. and Blikslager, A.T.}, year={2020}, month={Oct} }
 @article{ziegler_blikslager_2020, title={Sparing the gut: COX‐2 inhibitors herald a new era for treatment of horses with surgical colic}, url={https://doi.org/10.1111/eve.13189}, DOI={10.1111/eve.13189}, abstractNote={Summary Nonsteroidal anti‐inflammatory drugs ( NSAID s) are commonly used to manage a wide variety of conditions in horses, including management of colic. Flunixin meglumine is by far the most commonly used drug in the control of colic pain and inflammation and has become a go‐to for not only veterinarians but also horse‐owners and nonmedical equine professionals. NSAID use, however, has always been controversial in critical cases due to a high risk of adverse effects associated with their potent cyclo‐oxygenase ( COX ) inhibition. There are two important COX isoenzymes: COX ‐1 is generally beneficial for normal renal and gastrointestinal functions and COX ‐2 is associated with the pain and inflammation of disease. Newer selective NSAID s can target COX ‐2‐driven pathology while sparing important COX ‐1‐driven physiology, which is of critical importance in horses with severe gastrointestinal disease. Emerging research suggests that firocoxib, a COX ‐2‐selective NSAID labelled for use in horses, may be preferable for use in colic cases in spite of the decades‐long dogma that flunixin saves lives.}, journal={Equine Veterinary Education}, author={Ziegler, A. L. and Blikslager, A. T.}, year={2020}, month={Nov} }
 @article{erwin_touvron_odle_van landeghem_blikslager_ziegler_2020, title={iDISCO Allows Complete Visualization and Analysis of Postnatal Enteric Nervous System Development in a Comparative Pig Model}, volume={34}, ISSN={["1530-6860"]}, DOI={10.1096/fasebj.2020.34.s1.03991}, abstractNote={The enteric nervous system consists of a dense, complex network of neurons and glia which are instrumental in the maturation of normal intestinal physiology after birth. Our lab uses a comparative pig model to study the postnatal development of the enteric glial network and its role in regulating intestinal barrier functions in neonates. Immunolabeling‐enabled three‐dimensional (3D) imaging of solvent‐cleared organs (iDISCO) is a method of preparing tissue samples for volume imaging with a light sheet microscope. iDISCO has been optimized for use in primarily mouse organs and embryos for the study of early development and offers a more complete picture of the tissue than traditional histological analysis. Our objective was to optimize the iDISCO protocol for use in porcine intestinal tissue to allow complete qualitative and quantitative analysis of postnatal development of the enteric glial network in our comparative pig model. Antibodies against glial cell markers S100b, Sox10, and glial fibrillary acidic protein (GFAP) were used to triple‐stain fixed full‐thickness 3mm by 5mm samples of porcine jejunum using the iDISCO protocol. Samples were imaged with a light‐sheet microscope (Ultra‐II, LaVision BioTec ® ) using three different fluorescent channels and datasets were visualized and analyzed in 3D with Imaris software (Oxford Instruments ® ). The percent volume of GFAP + glial cells was quantified by manually masking individual intestinal villi and optimizing a surface algorithm to identify glial network structures within those villi. Antibodies against all three markers tested produced the predicted staining pattern with minimal non‐specific staining. Percent of jejunal villus volume occupied by GFAP + glia is higher in 6‐week‐old versus 2‐week‐old pig (0.49% versus 0.23%, *P≤0.05). Ongoing work will optimize quantification techniques for S100b and Sox10, and assess co‐localization patterns of these glial markers in the jejunum at discrete timepoints postnatally. iDISCO is a powerful imaging modality which will allow our lab to directly assess the expansion, complexity, and localization of glial cell subtypes by marker co‐expression analysis at discrete postnatal timepoints, and will be utilized in future studies to explore effects of disease and external interventions on the enteric glial network. Support or Funding Information NIH P30 DK034987 UNC CGIBD Pilot Feasibility; NIH T32 5T32DK007737‐22 UNC Basic Science GI Research Training Fellowship; USDA National Institute of Food and Agriculture, Animal Health Projects 1007263 and 07985; 2017 NC State CMI TPP Seed Grant}, number={S1}, journal={FASEB JOURNAL}, publisher={Wiley}, author={Erwin, Sara and Touvron, Melissa and Odle, Jack and Van Landeghem, Laurianne and Blikslager, Anthony and Ziegler, Amanda}, year={2020}, month={Apr} }
 @inproceedings{erwin_touvron_odle_landeghem_blikslager_ziegler_2020, place={San Diego, CA, USA}, title={iDISCO allows complete visualization and analysis of postnatal enteric nervous system development in a comparative pig model}, booktitle={Experimental Biology}, author={Erwin, S.E. and Touvron, M. and Odle, J. and Landeghem, L.Van and Blikslager, A.T. and Ziegler, A.L.}, year={2020}, month={Apr} }
 @article{ziegler_fogle_burke_blikslager_2019, title={Letter to the Editor: Bias in statistics or bias in equine veterinary medicine?}, volume={51}, ISSN={0425-1644 2042-3306}, url={http://dx.doi.org/10.1111/evj.13081}, DOI={10.1111/evj.13081}, abstractNote={See Correspondence by Freeman}, number={3}, journal={Equine Veterinary Journal}, publisher={Wiley}, author={Ziegler, A. L. and Fogle, C. A. and Burke, M. and Blikslager, A. T.}, year={2019}, month={Feb}, pages={423–423} }
 @inproceedings{ziegler_pridgen_landeghem_odle_magness_blikslager_2019, place={San Diego, CA, USA}, title={Rescue of Restitution Defect in a Neonatal Pig Intestinal Ischemia Model is associated with an Immature Enteric Glial Network}, booktitle={Digestive Disease Week}, author={Ziegler, A.L. and Pridgen, T.A. and Landeghem, L.Van and Odle, J. and Magness, S.T. and Blikslager, A.T.}, year={2019}, month={May} }
 @inproceedings{ziegler_pridgen_landeghem_odle_magness_blikslager_2019, place={Chicago, IL, USA}, title={Suckling piglets have a rescuable defect in intestinal barrier repair associated with an immature enteric glial network}, booktitle={Conference of Research Workers in Animal Disease}, author={Ziegler, A.L. and Pridgen, T.A. and Landeghem, L.Van and Odle, J. and Magness, S.T. and Blikslager, A.T.}, year={2019}, month={Nov} }
 @article{ziegler_pridgen_mills_gonzalez_landeghem_odle_blikslager_2018, title={Epithelial restitution defect in neonatal jejunum is rescued by juvenile mucosal homogenate in a pig model of intestinal ischemic injury and repair}, volume={7}, url={https://doi.org/10.1101/361352}, DOI={10.1101/361352}, abstractNote={ABSTRACT Intestinal ischemic injury results sloughing of the mucosal epithelium leading to host sepsis and death unless the mucosal barrier is rapidly restored. Neonatal necrotizing enterocolitis (NEC) and volvulus in infants is associated with intestinal ischemia, sepsis and high mortality rates. We have characterized intestinal ischemia/ repair using a highly translatable porcine model in which juvenile (6-8-week-old) pigs completely and efficiently restore barrier function by way of rapid epithelial restitution and tight junction re-assembly. In contrast, separate studies showed that younger neonatal (2-week-old) pigs exhibited less robust recovery of barrier function, which may model an important cause of high mortality rates in human infants with ischemic intestinal disease. Therefore, we aimed to further refine our repair model and characterize defects in neonatal barrier repair. Here we examine the defect in neonatal mucosal repair that we hypothesize is associated with hypomaturity of the epithelial and subepithelial compartments. Following jejunal ischemia in neonatal and juvenile pigs, injured mucosa was stripped from seromuscular layers and recovered ex vivo while monitoring transepithelial electrical resistance (TEER) and 3 H-mannitol flux as measures of barrier function. While ischemia-injured juvenile mucosa restored TEER above control levels, reduced flux over the recovery period and showed 93±4.7% wound closure, neonates exhibited no change in TEER, increased flux, and a 11±23.3% increase in epithelial wound size. Scanning electron microscopy revealed enterocytes at the wound margins of neonates failed to assume the restituting phenotype seen in restituting enterocytes of juveniles. To attempt rescue of injured neonatal mucosa, neonatal experiments were repeated with the addition of exogenous prostaglandins during ex vivo recovery, ex vivo recovery with full thickness intestine, in vivo recovery and direct application of injured mucosal homogenate from neonates or juveniles. Neither exogenous prostaglandins, intact seromuscular intestinal layers, nor in vivo recovery enhanced TEER or restitution in ischemia-injured neonatal mucosa. However, ex vivo exogenous application of injured juvenile mucosal homogenate produced a significant increase in TEER and enhanced histological restitution to 80±4.4% epithelial coveragein injured neonatal mucosa. Thus, neonatal mucosal repair can be rescued through direct contact with the cellular and non-cellular milieu of ischemia-injured mucosa from juvenile pigs. These findings support the hypothesis that a defect in mucosal repair in neonates is due to immature repair mechanisms within the mucosal compartment. Future studies to identify and rescue specific defects in neonatal intestinal repair mechanisms will drive development of novel clinical interventions to reduce mortality in infants affected by intestinal ischemic injury.}, publisher={Cold Spring Harbor Laboratory}, author={Ziegler, Amanda L. and Pridgen, Tiffany A. and Mills, Juliana K. and Gonzalez, Liara M. and Landeghem, Laurianne Van and Odle, Jack and Blikslager, Anthony T.}, year={2018}, month={Jul} }
 @article{ziegler_pridgen_mills_gonzalez_van landeghem_odle_blikslager_2018, title={Epithelial restitution defect in neonatal jejunum is rescued by juvenile mucosal homogenate in a pig model of intestinal ischemic injury and repair}, volume={13}, ISSN={1932-6203}, url={http://dx.doi.org/10.1371/journal.pone.0200674}, DOI={10.1371/journal.pone.0200674}, abstractNote={Intestinal ischemic injury results sloughing of the mucosal epithelium leading to host sepsis and death unless the mucosal barrier is rapidly restored. Volvulus and neonatal necrotizing enterocolitis (NEC) in infants have been associated with intestinal ischemia, sepsis and high mortality rates. We have characterized intestinal ischemia/repair using a highly translatable porcine model in which juvenile (6-8-week-old) pigs completely and efficiently restore barrier function by way of rapid epithelial restitution and tight junction re-assembly. In contrast, separate studies showed that younger neonatal (2-week-old) pigs exhibited less robust recovery of barrier function, which may model an important cause of high mortality rates in human infants with ischemic intestinal disease. Therefore, we aimed to further refine our repair model and characterize defects in neonatal barrier repair. Here we examine the defect in neonatal mucosal repair that we hypothesize is associated with hypomaturity of the epithelial and subepithelial compartments. Following jejunal ischemia in neonatal and juvenile pigs, injured mucosa was stripped from seromuscular layers and recovered ex vivo while monitoring transepithelial electrical resistance (TEER) and 3H-mannitol flux as measures of barrier function. While ischemia-injured juvenile mucosa restored TEER above control levels, reduced flux over the recovery period and showed 93±4.7% wound closure, neonates exhibited no change in TEER, increased flux, and a 11±23.3% increase in epithelial wound size. Scanning electron microscopy revealed enterocytes at the wound margins of neonates failed to assume the restituting phenotype seen in restituting enterocytes of juveniles. To attempt rescue of injured neonatal mucosa, neonatal experiments were repeated with the addition of exogenous prostaglandins during ex vivo recovery, ex vivo recovery with full thickness intestine, in vivo recovery and direct application of injured mucosal homogenate from neonates or juveniles. Neither exogenous prostaglandins, intact seromuscular intestinal layers, nor in vivo recovery enhanced TEER or restitution in ischemia-injured neonatal mucosa. However, ex vivo exogenous application of injured juvenile mucosal homogenate produced a significant increase in TEER and enhanced histological restitution to 80±4.4% epithelial coverage in injured neonatal mucosa. Thus, neonatal mucosal repair can be rescued through direct contact with the cellular and non-cellular milieu of ischemia-injured mucosa from juvenile pigs. These findings support the hypothesis that a defect in mucosal repair in neonates is due to immature repair mechanisms within the mucosal compartment. Future studies to identify and rescue specific defects in neonatal intestinal repair mechanisms will drive development of novel clinical interventions to reduce mortality in infants affected by intestinal ischemic injury.}, number={8}, journal={PLOS ONE}, publisher={Public Library of Science (PLoS)}, author={Ziegler, Amanda L. and Pridgen, Tiffany A. and Mills, Juliana K. and Gonzalez, Liara M. and Van Landeghem, Laurianne and Odle, Jack and Blikslager, Anthony T.}, editor={Karhausen, JörnEditor}, year={2018}, month={Aug}, pages={e0200674} }
 @article{ziegler_freeman_fogle_burke_davis_cook_southwood_blikslager_2018, title={Multicentre, blinded, randomised clinical trial comparing the use of flunixin meglumine with firocoxib in horses with small intestinal strangulating obstruction}, volume={51}, ISSN={0425-1644 2042-3306}, url={http://dx.doi.org/10.1111/evj.13013}, DOI={10.1111/evj.13013}, abstractNote={Summary Background Small intestinal strangulating obstruction (SISO) is associated with endotoxaemia which leads to an increased risk of death. Nonsteroidal anti‐inflammatory drugs (NSAIDs) are used to treat signs of endotoxaemia by inhibiting cyclo‐oxygenases (COX). COX‐1 is expressed constitutively and promotes gut barrier function, whereas COX‐2 is inducible and contributes to the signs of endotoxaemia. In preclinical SISO trials, intestinal barrier recovery was more complete with reductions in endotoxin permeability in horses treated with COX‐2 selective NSAIDs as compared with horses treated with flunixin meglumine. Objectives We hypothesised that treatment of post‐surgical SISO horses with firocoxib (COX‐2 selective) would reduce the signs of endotoxaemia to a greater extent than flunixin meglumine (nonselective COX inhibitor) while continuing to provide similar levels of pain control. Study design Blinded randomised clinical trial. Methods In addition to clinical monitoring, preoperative and 12‐, 24‐ and 48‐h post‐operative plasma samples were assessed for prostaglandin E 2 (PGE 2 ), thromboxane B 2 (TXB 2 ), TNF⍺ and soluble CD14 (sCD14). Results In 56 recruited SISO horses, either flunixin meglumine (1.1 mg/kg, i.v., q12h) or firocoxib (0.3 mg/kg, i.v. loading dose; 0.1 mg/kg, i.v., q24h) was given in the post‐operative period in three university hospitals from 2015 to 2017. COX‐2 selectivity was confirmed by a relative lack of inhibition of the COX‐1 prostanoid TXB 2 by firocoxib and significant inhibition by flunixin meglumine (P = 0.014). Both drugs inhibited the COX‐2 prostanoid PGE 2 . There were no significant differences in pain scores between groups (P = 0.2). However, there was a 3.23‐fold increased risk (P = 0.04) of increased plasma sCD14 in horses treated with flunixin meglumine, a validated biomarker of equine endotoxaemia. Main limitations Horses were all treated with flunixin meglumine prior to referral. In addition, many horses were treated with lidocaine, which has been shown to mitigate the deleterious effects of flunixin meglumine. Conclusions In SISO cases, firocoxib reduced a biomarker of endotoxaemia as compared with flunixin meglumine while continuing to provide similar levels of pain control.}, number={3}, journal={Equine Veterinary Journal}, publisher={Wiley}, author={Ziegler, A. L. and Freeman, C. K. and Fogle, C. A. and Burke, M. J. and Davis, J. L. and Cook, V. L. and Southwood, L. L. and Blikslager, A. T.}, year={2018}, month={Sep}, pages={329–335} }
 @inproceedings{ziegler_pridgen_landeghem_gonzalez_odle_blikslager_2018, place={College Station, TX, USA}, title={Neonatal Defect in Subacute Barrier Repair is Rescued by Juvenile Mucosal Homogenate in a Pig Model of Intestinal Ischemia and Repair}, volume={Aug 3}, booktitle={National Veterinary Scholars Symposium}, author={Ziegler, A.L. and Pridgen, T.A. and Landeghem, L.Van and Gonzalez, L.M. and Odle, J. and Blikslager, A.T.}, year={2018}, month={Aug} }
 @inproceedings{ziegler_freeman_fogle_burke_davis_southwood_cook_blikslager_2018, place={Oral Abstract}, title={Randomized control trial comparing flunixin meglumine and firocoxib in equine strangulating obstruction}, booktitle={American Association of Equine Practitioners Convention}, publisher={American Association of Equine Practitioners}, author={Ziegler, A.L. and Freeman, C.K. and Fogle, C.A. and Burke, M.J. and Davis, J. and Southwood, L.L. and Cook, V.L. and Blikslager, A.T.}, year={2018}, month={Dec} }
 @inproceedings{ziegler_pridgen_landeghem_gonzalez_odle_blikslager_2017, place={Lexington, KY, USA}, title={Neonates have a reduced ability to repair jejunal mucosal injury as compared to weanlings in a porcine model of small intestinal strangulating obstruction}, booktitle={12th International Colic Research Symposium}, author={Ziegler, A.L. and Pridgen, T.A. and Landeghem, L.Van and Gonzalez, L.M. and Odle, J. and Blikslager, A.T.}, year={2017}, month={Jul} }
 @inproceedings{ziegler_pridgen_landeghem_gonzalez_odle_blikslager_2017, place={CO, USA}, title={Neonates have reduced epithelial wound healing associated with fewer enteric glial cells as compared to juveniles in a pig model of intestinal ischemic injury and repair}, booktitle={FASEB Gastrointestinal Tract XVII}, author={Ziegler, A.L. and Pridgen, T.A. and Landeghem, L.Van and Gonzalez, L.M. and Odle, J. and Blikslager, A.T.}, year={2017}, month={Jul} }
 @inproceedings{ziegler_fogle_burke_davis_southwood_cook_blikslager_2017, place={Lexington, KY, USA}, title={Randomized control trial comparing flunixin and firocoxib in equine strangulating obstruction}, booktitle={12th International Colic Research Symposium}, author={Ziegler, A.L. and Fogle, C. and Burke, M. and Davis, J. and Southwood, L. and Cook, V. and Blikslager, A.T.}, year={2017}, month={Jul} }
 @article{ziegler_fogle_blikslager_2017, title={Update on the use of cyclooxygenase-2-selective nonsteroidal anti-inflammatory drugs in horses}, volume={250}, ISSN={0003-1488}, url={http://dx.doi.org/10.2460/javma.250.11.1271}, DOI={10.2460/javma.250.11.1271}, abstractNote={Nonsteroidal anti-inflammatory drugs work through inhibition of cyclooxygenase (COX) and are highly effective for the treatment of pain and inflammation in horses. There are 2 clinically relevant isoforms of COX. Cyclooxygenase-1 is constitutively expressed and is considered important for a variety of physiologic functions, including gastrointestinal homeostasis. Thus, NSAIDs that selectively inhibit COX-2 while sparing COX-1 may be associated with a lower incidence of adverse gastrointestinal effects. Various formulations of firocoxib, a COX-2-selective NSAID, labeled for use in horses are available in the United States. Equine practitioners should know that the FDA limits the use of firocoxib to formulations labeled for horses, regardless of price concerns. In addition, practitioners will benefit from understanding the nuances of firocoxib administration, including the importance of correct dosing and the contraindications of combining NSAIDs. Together with knowledge of the potential advantages of COX-2 selectivity, these considerations will help veterinarians select and treat patients that could benefit from this new class of NSAID.}, number={11}, journal={Journal of the American Veterinary Medical Association}, publisher={American Veterinary Medical Association (AVMA)}, author={Ziegler, Amanda and Fogle, Callie and Blikslager, Anthony}, year={2017}, month={Jun}, pages={1271–1274} }
 @article{ziegler_gonzalez_blikslager_2016, title={Large Animal Models: The Key to Translational Discovery in Digestive Disease Research}, volume={2}, ISSN={2352-345X}, url={http://dx.doi.org/10.1016/j.jcmgh.2016.09.003}, DOI={10.1016/j.jcmgh.2016.09.003}, abstractNote={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 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.}, number={6}, journal={Cellular and Molecular Gastroenterology and Hepatology}, publisher={Elsevier BV}, author={Ziegler, Amanda and Gonzalez, Liara and Blikslager, Anthony}, year={2016}, month={Nov}, pages={716–724} }
 @inproceedings{ziegler_pridgen_carnighan_odle_gonzalez_blikslager_2016, place={San Diego, CA, USA}, title={Neonates Have a Reduced Ability to Repair Jejunal Mucosal Injury as Compared to Juveniles in a Pig Model of Ischemia/ Reperfusion Injury}, booktitle={Digestive Disease Week}, author={Ziegler, A.L. and Pridgen, T.A. and Carnighan, G. and Odle, J. and Gonzalez, L.M. and Blikslager, A.T.}, year={2016}, month={May} }
 @article{newman_sills_hanrahan_ziegler_tidd_cook_sannes_2015, title={Expression of WNT5A in Idiopathic Pulmonary Fibrosis and Its Control by TGF-β and WNT7B in Human Lung Fibroblasts}, volume={64}, ISSN={0022-1554 1551-5044}, url={http://dx.doi.org/10.1369/0022155415617988}, DOI={10.1369/0022155415617988}, abstractNote={The wingless (Wnt) family of signaling ligands contributes significantly to lung development and is highly expressed in patients with usual interstitial pneumonia (UIP). We sought to define the cellular distribution of Wnt5A in the lung tissue of patients with idiopathic pulmonary fibrosis (IPF) and the signaling ligands that control its expression in human lung fibroblasts and IPF myofibroblasts. Tissue sections from 40 patients diagnosed with IPF or UIP were probed for the immunolocalization of Wnt5A. Further, isolated lung fibroblasts from normal or IPF human lungs, adenovirally transduced for the overexpression or silencing of Wnt7B or treated with TGF-β1 or its inhibitor, were analyzed for Wnt5A protein expression. Wnt5A was expressed in IPF lungs by airway and alveolar epithelium, smooth muscle cells, endothelium, and myofibroblasts of fibroblastic foci and throughout the interstitium. Forced overexpression of Wnt7B with or without TGF-β1 treatment significantly increased Wnt5A protein expression in normal human smooth muscle cells and fibroblasts but not in IPF myofibroblasts where Wnt5A was already highly expressed. The results demonstrate a wide distribution of Wnt5A expression in cells of the IPF lung and reveal that it is significantly increased by Wnt7B and TGF-β1, which, in combination, could represent key signaling pathways that modulate the pathogenesis of IPF.}, number={2}, journal={Journal of Histochemistry & Cytochemistry}, publisher={SAGE Publications}, author={Newman, Donna R. and Sills, W. Shane and Hanrahan, Katherine and Ziegler, Amanda and Tidd, Kathleen McGinnis and Cook, Elizabeth and Sannes, Philip L.}, year={2015}, month={Nov}, pages={99–111} }
 @inproceedings{welch_newman_sannes_2013, place={Lansing, MI, USA}, title={Porcine alveolar type II cells as a potential model for human alveolar epithelium in culture.}, booktitle={National Veterinary Scholars Symposium}, author={Welch, A.L. and Newman, D.R. and Sannes, P.L.}, year={2013}, month={Aug} }