@article{stewart_kopper_mckinney-aguirre_veerasamy_sahoo_freund_gonzalez_2024, title={Assessment of equine intestinal epithelial junctional complexes and barrier permeability using a monolayer culture system}, volume={11}, ISSN={["2297-1769"]}, DOI={10.3389/fvets.2024.1455262}, abstractNote={Gastrointestinal disease is a leading cause of death in mature horses. A lack of in vitro modeling has impeded the development of novel therapeutics. The objectives of this study were to develop and further characterize a small intestinal monolayer cell culture derived from equine jejunum including establishing normal measurements of intestinal permeability and restitution. Three-dimensional enteroids, derived from postmortem sampling of equine jejunum, were utilized to develop confluent epithelial monolayers. The presence of differentiated intestinal epithelial cell types and tight junctions were confirmed using histology, reverse transcription PCR (RT-PCR), RNAscope, protein immunofluorescence and transmission electron microscopy. Transepithelial resistance (TER) and macromolecule flux were assessed as measurements of paracellular and transcellular permeability. Scratch assays were utilized to model and assess intestinal restitution. Monolayer cell cultures reached 100% confluency by ~5–7 days. Equine jejunum monolayers were confirmed as epithelial in origin, with identification of differentiated intestinal epithelial cell types and evidence of tight junction proteins. Function of the intestinal barrier was supported by acquisition of physiologically normal TER values (179.9 ± 33.7 ohms*cm 2 ) and limited macromolecule flux (22 ± 8.8% at 60 min). Additionally, following a scratch wound, epithelial cell monolayers migrated to close gap defects within 24 h. In conclusion, this study describes the development of a novel intestinal epithelial monolayer cell culture for equine jejunum, and provides evidence of intestinal epithelial cell differentiation, formation of physiologically relevant barrier function and use as a model of intestinal restitution to test potential therapeutics for equine colic.}, journal={FRONTIERS IN VETERINARY SCIENCE}, author={Stewart, Amy Stieler and Kopper, Jamie J. and McKinney-Aguirre, Caroline and Veerasamy, Brittany and Sahoo, Dipak Kumar and Freund, John M. and Gonzalez, Liara M.}, year={2024}, month={Oct} }
 @article{ludwig_abraham_mckinney_freund_stewart_garman_barbas_sudan_gonzalez_2023, title={45: Comparison of the Effects of Normothermic Machine Perfusion and Cold Storage Preservation on Porcine Intestinal Allograft Regenerative Potential and Viability}, volume={107}, ISSN={0041-1337}, url={http://dx.doi.org/10.1097/01.tp.0000945636.34372.db}, DOI={10.1097/01.tp.0000945636.34372.db}, abstractNote={Historically, intestinal transplantation (IT) has been reserved as the last treatment option for patients with irreversible intestinal failure who are unable to tolerate total parenteral nutrition. Successful IT is reliant upon graft health at the time of donation, minimizing graft injury that may occur during procurement, storage, and IT, and the ability of the graft to heal following insult. Unfortunately, the intestine is easily damaged by ischemia-reperfusion injury (IRI). IRI induces intestinal epithelial cell apoptosis and damages the mucosal barrier, which can result in bacterial translocation and activation of the local and systemic immune and inflammatory response, ultimately contributing to graft failure, rejection, and decreased recipient survival. The current, preferred method of intestinal preservation prior to IT is static cold storage (CS), however the prolonged hypothermic ischemia of CS causes cell injury and intensifies the IRI that occurs during transplantation. Furthermore, IRI to the epithelial crypt region diminishes the intestine’s ability to heal by inducing loss of the highly proliferative intestinal stem cells (ISCs) that are responsible for maintenance, regeneration, and repair of the epithelium, critical to graft health. Thus, the investigation of alternative organ preservation techniques that reduce IRI, cellular damage, and graft injury are warranted to overall improve IT success. Normothermic machine perfusion (NMP) is a preservation method that reduces inflammation and promotes graft regeneration in other organs by preventing CS-associated IRI. However, NMP has not been described for intestine. We hypothesized that, compared to CS, intestinal NMP will induce less epithelial injury and better protect ISC regenerative potential and viability. 15 porcine intestines were flushed with UW solution, stored at 4°C (CS), or perfused with 34°C perfusate (NMP) for 6hr, and transplanted (n=9). Recipient pigs were recovered from anesthesia. Jejunal and ileal segments were collected immediately after flushing, serving as control tissue (CO), after 6hr of CS or NMP, and after 1hr of reperfusion post-IT. Histologic injury was assessed. Crypts isolated after flushing (CO), 6hr CS or NMP, and 1hr of reperfusion post-IT were cultured. Spheroid number, size, and EdU staining quantified ISC viability and proliferation. Expression of ISC and cellular proliferation genes and proteins were measured. Histologically, NMP tissue had mild epithelial erosion and increased columnar cell attenuation and expression of ISC and proliferation genes/proteins was observed. NMP spheroid areas and proliferating cell numbers were significantly larger than control and CS. Apoptotic cells were increased following CS. Post-graft reperfusion, CS had increased injury compared to uninjured control and NMP tissue. Compared to CS, NMP may improve graft regenerative potential, resulting in transplantation of healthier bowel and superior recipient survival.}, number={7S}, journal={Transplantation}, publisher={Ovid Technologies (Wolters Kluwer Health)}, author={Ludwig, Elsa and Abraham, Nader and McKinney, Caroline and Freund, John and Stewart, Amy and Garman, Katherine and Barbas, Andrew and Sudan, Debra and Gonzalez, Liara}, year={2023}, month={Jun}, pages={25–25} }
 @article{schaaf_polkoff_carter_stewart_sheahan_freund_ginzel_snyder_roper_piedrahita_et al._2023, title={A LGR5 reporter pig model closely resembles human intestine for improved study of stem cells in disease}, volume={37}, ISSN={["1530-6860"]}, DOI={10.1096/fj.202300223R}, abstractNote={Abstract Intestinal epithelial stem cells (ISCs) are responsible for intestinal epithelial barrier renewal; thereby, ISCs play a critical role in intestinal pathophysiology research. While transgenic ISC reporter mice are available, advanced translational studies lack a large animal model. This study validates ISC isolation in a new porcine Leucine Rich Repeat Containing G Protein‐Coupled Receptor 5 (LGR5) reporter line and demonstrates the use of these pigs as a novel colorectal cancer (CRC) model. We applied histology, immunofluorescence, fluorescence‐activated cell sorting, flow cytometry, gene expression quantification, and 3D organoid cultures to whole tissue and single cells from the duodenum, jejunum, ileum, and colon of LGR5‐H2B‐GFP and wild‐type pigs. Ileum and colon LGR5‐H2B‐GFP, healthy human, and murine biopsies were compared by mRNA fluorescent in situ hybridization (FISH). To model CRC, adenomatous polyposis coli ( APC ) mutation was induced by CRISPR/Cas9 editing in porcine LGR5‐H2B‐GFP colonoids. Crypt‐base, green fluorescent protein (GFP) expressing cells co‐localized with ISC biomarkers. LGR5‐H2B‐GFP hi cells had significantly higher LGR5 expression ( p < .01) and enteroid forming efficiency ( p < .0001) compared with LGR5‐H2B‐GFP med/lo/neg cells. Using FISH, similar LGR5, OLFM4, HOPX, LYZ , and SOX9 expression was identified between human and LGR5‐H2B‐GFP pig crypt‐base cells. LGR5‐H2B‐GFP/ APC null colonoids had cystic growth in WNT/R‐spondin‐depleted media and significantly upregulated WNT/β‐catenin target gene expression ( p < .05). LGR5 + ISCs are reproducibly isolated in LGR5‐H2B‐GFP pigs and used to model CRC in an organoid platform. The known anatomical and physiologic similarities between pig and human, and those shown by crypt‐base FISH, underscore the significance of this novel LGR5‐H2B‐GFP pig to translational ISC research.}, number={6}, journal={FASEB JOURNAL}, author={Schaaf, Cecilia R. and Polkoff, Kathryn M. and Carter, Amber and Stewart, Amy S. and Sheahan, Breanna and Freund, John and Ginzel, Joshua and Snyder, Joshua C. and Roper, Jatin and Piedrahita, Jorge A. and et al.}, year={2023}, month={Jun} }
 @article{veerasammy_gonzalez_báez‐ramos_schaaf_stewart_ludwig_mckinney‐aguirre_freund_robertson_gonzalez_2023, title={Changes in equine intestinal stem/progenitor cell number at resection margins in cases of small intestinal strangulation}, volume={55}, ISSN={0425-1644 2042-3306}, url={http://dx.doi.org/10.1111/evj.13927}, DOI={10.1111/evj.13927}, abstractNote={Abstract Background Intestinal epithelial stem cells (ISC) are responsible for epithelial regeneration and are critical to the intestine's ability to regain barrier function following injury. Evaluating ISC biomarker expression in cases of small intestinal strangulation (SIS) may provide insight into clinical progression. Objectives Intestinal resection margins from cases of SIS were evaluated to determine if (1) evidence of injury could be identified using histomorphometry, (2) ISC biomarker expression was decreased in the proximal resection margin compared to control and distal resection margin, and (3) the ISC biomarker expression was associated with the number of preoperative risk factors negatively related to outcome, post‐operative complications, or case outcome. Study design Retrospective cohort study. Methods Intestinal samples were obtained intraoperatively from resection margins of adult horses with SIS and horses euthanised for reasons unrelated to colic. Preoperative risk factors negatively related to outcome, post‐operative complications, and case outcome were obtained from medical records. Horses were grouped as euthanised intraoperatively, postoperatively, or survived to discharge. Histomorphometry and immunofluorescence were performed to evaluate tissue architecture and ISC and progenitor cell number. Groups were compared using one‐way ANOVA. Associations between biomarker expression and the number of preoperative risk factors and post‐operative complications negatively related to outcome were determined using linear regression modelling. Results Thirty‐six cases of SIS were evaluated. Ki67 + cell counts were decreased in the proximal (mean = 15.45 cells; 95% CI = 10.27–20.63; SD = 4.17; p = 0.02) and distal resection margins (mean = 15.05; 95% CI = 8.46–21.64; SD = 4.141; p = 0.03) in horses euthanised postoperatively compared to control (mean = 23.62 cells; 95% CI = 19.42–27.83; SD = 5.883). In the distal resection margin, an increase in SOX9 + Ki67 + cells were associated with a decrease in the total number of preoperative risk factors negatively related to outcome (95% CI = 0.236–1.123; p = 0.008, SE = 0.1393). Main limitations Small population size. Conclusions Proliferating cell and ISC numbers may be associated with case outcome.}, number={6}, journal={Equine Veterinary Journal}, publisher={Wiley}, author={Veerasammy, Brittany and Gonzalez, Gabriel and Báez‐Ramos, Patricia and Schaaf, Cecilia R. and Stewart, Amy Stieler and Ludwig, Elsa K. and McKinney‐Aguirre, Caroline and Freund, John and Robertson, James and Gonzalez, Liara M.}, year={2023}, month={Feb}, pages={995–1002} }
 @article{ludwig_abraham_schaaf_mckinney_freund_stewart_veerasammy_thomas_cardona_garman_et al._2023, title={Comparison of the effects of normothermic machine perfusion and cold storage preservation on porcine intestinal allograft regenerative potential and viability}, volume={24}, ISSN={1600-6135}, url={http://dx.doi.org/10.1016/j.ajt.2023.10.026}, DOI={10.1016/j.ajt.2023.10.026}, abstractNote={Intestinal transplantation (IT) is the final treatment option for intestinal failure. Static cold storage (CS) is the standard preservation method used for intestinal allografts. However, CS and subsequent transplantation induce ischemia-reperfusion injury (IRI). Severe IRI impairs epithelial barrier function, including loss of intestinal stem cells (ISC), critical to epithelial regeneration. Normothermic machine perfusion (NMP) preservation of kidney and liver allografts minimizes CS-associated IRI; however, it has not been used clinically for IT. We hypothesized that intestine NMP would induce less epithelial injury and better protect the intestine's regenerative ability when compared with CS. Full-length porcine jejunum and ileum were procured, stored at 4 °C, or perfused at 34 °C for 6 hours (T6), and transplanted. Histology was assessed following procurement (T0), T6, and 1 hour after reperfusion. Real-time quantitative reverse transcription polymerase chain reaction, immunofluorescence, and crypt culture measured ISC viability and proliferative potential. A greater number of NMP-preserved intestine recipients survived posttransplant, which correlated with significantly decreased tissue injury following 1-hour reperfusion in NMP compared with CS samples. Additionally, ISC gene expression, spheroid area, and cellular proliferation were significantly increased in NMP-T6 compared with CS-T6 intestine. NMP appears to reduce IRI and improve graft regeneration with improved ISC viability and proliferation.}, number={4}, journal={American Journal of Transplantation}, publisher={Elsevier BV}, author={Ludwig, Elsa K. and Abraham, Nader and Schaaf, Cecilia R. and McKinney, Caroline A. and Freund, John and Stewart, Amy S. and Veerasammy, Brittany A. and Thomas, Mallory and Cardona, Diana M. and Garman, Katherine and et al.}, year={2023}, month={Oct}, pages={564–576} }
 @article{stewart_schaaf_veerasammy_freund_gonzalez_2022, title={Culture of equine intestinal epithelial stem cells after delayed tissue storage for future applications}, volume={18}, ISSN={["1746-6148"]}, DOI={10.1186/s12917-022-03552-6}, abstractNote={Equine intestinal epithelial stem cells (ISCs) serve as potential targets to treat horses with severe intestinal injury. The ability to isolate and store ISCs from intestinal biopsies creates an opportunity for both in vitro experiments to study ISC dynamics in a variety of intestinal diseases, and, in the future, utilize these cells as a possible therapy. If biopsies could be successfully stored prior to processing for ISCs, this would increase the availability of sample repositories for future experimental and therapeutic use. However, delayed culture of equine ISCs following prolonged sample storage has not been described. The objective of this study was to describe the isolation and culture of equine ISCs following delayed tissue storage. Small intestinal full thickness biopsies were collected post euthanasia. Fresh tissue was immediately processed or stored at 4 °C for 24, 48 and 72 h (H) before processing. Intestinal stem cells (crypts) were dissociated and cultured. Size, growth efficiency and proliferation potential were compared between resultant enteroids ("mini-guts") derived from each storage timepoint. In a separate study, growth efficiency of cryopreserved crypts was compared to cryopreserved enteroid fragments to investigate prolonged storage techniques.Intestinal crypts were successfully isolated and cultured from all timepoints. At 72H post initial collection, the intestine was friable with epithelial sloughing; resultant dissociation yielded more partial crypts. Enteroids grown from crypts isolated at 72H were smaller with less proliferative potential (bud units, (median 6.5, 3.75-14.25)) than control (median 25, 15-28, p < 0.0001). No statistical differences were noted from tissues stored for 24H compared to control. Following cryopreservation, growth efficiency improved when cells were stored as enteroid fragments (median 81.6%, 66.2-109) compared to crypts (median 21.2%, 20-21.5, p = 0.01). The main limitations included a small sample size and lack of additional functional assays on enteroids.Equine ISCs can be isolated and cultured after prolonged tissue storage. Resultant enteroids had minimal differences even after 24-48H of whole tissue storage. This suggests that ISCs could be isolated for several days from samples properly stored after procedures, including surgery or necropsy, and used to create ISC repositories for study or therapy of equine intestinal diseases.}, number={1}, journal={BMC VETERINARY RESEARCH}, author={Stewart, Amy Stieler and Schaaf, Cecilia R. and Veerasammy, Brittany and Freund, John M. and Gonzalez, Liara M.}, year={2022}, month={Dec} }
 @article{abraham_ludwig_schaaf_veerasammy_stewart_mckinney_freund_brassil_samy_gao_et al._2022, title={Orthotopic Transplantation of the Full-length Porcine Intestine After Normothermic Machine Perfusion}, volume={8}, ISSN={2373-8731}, url={http://dx.doi.org/10.1097/TXD.0000000000001390}, DOI={10.1097/TXD.0000000000001390}, abstractNote={Background. Successful intestinal transplantation is currently hindered by graft injury that occurs during procurement and storage, which contributes to postoperative sepsis and allograft rejection. Improved graft preservation may expand transplantable graft numbers and enhance posttransplant outcomes. Superior transplant outcomes have recently been demonstrated in clinical trials using machine perfusion to preserve the liver. We hypothesized that machine perfusion preservation of intestinal allografts could be achieved and allow for transplantation in a porcine model. Methods. Using a translational porcine model, we developed a device for intestinal perfusion. Intestinal samples were collected at the time of organ procurement, and after 6 h of machine perfusion for gross and histologic evaluation, hourly chemistry panels were performed on the perfusate and were used for protocol optimization. Following transplantation, porcine recipient physical activity, systemic blood parameters, and vital signs were monitored for 2 d before sacrifice. Results. In initial protocol development (generation 1, n = 8 grafts), multiple metabolic, electrolyte, and acid-base derangements were measured. These factors coincided with graft and mesenteric edema and luminal hemorrhage and were addressed with the addition of dialysis. In the subsequent protocol (generation 2, n = 9 grafts), differential jejunum and ileum perfusion were observed resulting in gross evidence of ileal ischemia. Modifications in vasodilating medications enhanced ileal perfusion (generation 3, n = 4 grafts). We report successful transplantation of 2 porcine intestinal allografts after machine perfusion with postoperative clinical and gross evidence of normal gut function. Conclusions. This study reports development and optimization of machine perfusion preservation of small intestine and successful transplantation of intestinal allografts in a porcine model.}, number={11}, journal={Transplantation Direct}, publisher={Ovid Technologies (Wolters Kluwer Health)}, author={Abraham, Nader and Ludwig, Elsa K. and Schaaf, Cecilia R. and Veerasammy, Brittany and Stewart, Amy S. and McKinney, Caroline and Freund, John and Brassil, John and Samy, Kannan P. and Gao, Qimeng and et al.}, year={2022}, month={Oct}, pages={e1390} }
 @article{stewart_schaaf_luff_freund_becker_tufts_robertson_gonzalez_2021, title={HOPX+ injury-resistant intestinal stem cells drive epithelial recovery after severe intestinal ischemia}, volume={321}, ISSN={["1522-1547"]}, url={https://doi.org/10.1152/ajpgi.00165.2021}, DOI={10.1152/ajpgi.00165.2021}, abstractNote={Intestinal ischemia is a life-threatening emergency with mortality rates of 50%-80% due to epithelial cell death and resultant barrier loss. Loss of the epithelial barrier occurs in conditions including intestinal volvulus and neonatal necrotizing enterocolitis. Survival depends on effective epithelial repair; crypt-based intestinal epithelial stem cells (ISCs) are the source of epithelial renewal in homeostasis and after injury. Two ISC populations have been described:}, number={5}, journal={AMERICAN JOURNAL OF PHYSIOLOGY-GASTROINTESTINAL AND LIVER PHYSIOLOGY}, publisher={American Physiological Society}, author={Stewart, Amy Stieler and Schaaf, Cecilia Renee and Luff, Jennifer A. and Freund, John M. and Becker, Thomas C. and Tufts, Sara R. and Robertson, James B. and Gonzalez, Liara M.}, year={2021}, month={Oct}, pages={G588–G602} }
 @article{steward_hassel_martin_doddman_stewart_elzer_southwood_2020, title={Geographic Disparities in Clinical Characteristics of Duodenitis–Proximal Jejunitis in Horses in the United States}, volume={93}, url={https://doi.org/10.1016/j.jevs.2020.103192}, DOI={10.1016/j.jevs.2020.103192}, abstractNote={Duodenitis–proximal jejunitis (DPJ) is an idiopathic and potentially fatal disease of horses characterized by abdominal pain, proximal intestinal inflammation, and subsequent gastric and small intestinal fluid accumulation. Although this disease is known to be costly and life threatening in the equine industry, the severity of clinical signs can vary widely, and an exact etiology has yet to be elucidated. This study looked to identify differences in clinical parameters of horses with DPJ between geographic regions in an effort to corroborate anecdotal reports and support theories of differing etiologies. Case records were compared from veterinary academic referral hospitals in three different geographic locations in the United States to determine if significant differences in clinical, clinicopathologic, and prognostic characteristics exist among horses with DPJ. Clinical measurements on presentation that were significantly different between regions included heart rate, peritoneal total protein, albumin, anion gap, aspartate aminotransferase, gamma-glutamyl transferase, sodium, chloride, potassium, and creatinine. Duration of hospitalization and maximum body temperature while hospitalized were also different between regions. There were no significant differences in peritoneal cell count, total white blood cell count, neutrophil count, band neutrophils, calcium, total plasma protein, temperature on presentation, duration of reflux, total reflux volume, or age between hospitals. The mortality rates between hospitals were not significantly different. Increased severity of clinical signs and biochemical abnormalities were identified in the Southeastern United States hospital compared with the Northeastern and Western hospitals. A prospective, multicenter case–control study could identify risk factors contributing toward regional differences in this disease in the future.}, journal={Journal of Equine Veterinary Science}, publisher={Elsevier BV}, author={Steward, Sara K.T. and Hassel, Diana M. and Martin, Holly and Doddman, Courtney and Stewart, Amy and Elzer, Elizabeth J. and Southwood, Louise L.}, year={2020}, month={Oct}, pages={103192} }
 @article{gonzalez_stewart_freund_kucera_dekaney_magness_blikslager_2019, title={Preservation of reserve intestinal epithelial stem cells following severe ischemic injury}, volume={316}, ISSN={0193-1857 1522-1547}, url={http://dx.doi.org/10.1152/ajpgi.00262.2018}, DOI={10.1152/ajpgi.00262.2018}, abstractNote={Intestinal ischemia is an abdominal emergency with a mortality rate >50%, leading to epithelial barrier loss and subsequent sepsis. Epithelial renewal and repair after injury depend on intestinal epithelial stem cells (ISC) that reside within the crypts of Lieberkühn. Two ISC populations critical to epithelial repair have been described: 1) active ISC (aISC; highly proliferative; leucine-rich-repeat-containing G protein-coupled receptor 5 positive, sex determining region Y-box 9 positive) and 2) reserve ISC [rISC; less proliferative; homeodomain only protein X (Hopx)+]. Yorkshire crossbred pigs (8-10 wk old) were subjected to 1-4 h of ischemia and 1 h of reperfusion or recovery by reversible mesenteric vascular occlusion. This study was designed to evaluate whether ISC-expressing biomarkers of aISCs or rISCs show differential resistance to ischemic injury and different contributions to the subsequent repair and regenerative responses. Our data demonstrate that, following 3-4 h ischemic injury, aISC undergo apoptosis, whereas rISC are preserved. Furthermore, these rISC are retained ex vivo in spheroids in which cell populations are enriched in the rISC biomarker Hopx. These cells appear to go on to provide a proliferative pool of cells during the recovery period. Taken together, these data indicate that Hopx+ cells are resistant to injury and are the likely source of epithelial renewal following prolonged ischemic injury. It is therefore possible that targeting reserve stem cells will lead to new therapies for patients with severe intestinal injury. NEW & NOTEWORTHY The population of reserve less-proliferative intestinal epithelial stem cells appears resistant to injury despite severe epithelial cell loss, including that of the active stem cell population, which results from prolonged mesenteric ischemia. These cells can change to an activated state and are likely indispensable to regenerative processes. Reserve stem cell targeted therapies may improve treatment and outcome of patients with ischemic disease.}, number={4}, journal={American Journal of Physiology-Gastrointestinal and Liver Physiology}, publisher={American Physiological Society}, author={Gonzalez, Liara M. and Stewart, Amy Stieler and Freund, John and Kucera, Cecilia Renee and Dekaney, Christopher M. and Magness, Scott T. and Blikslager, Anthony T.}, year={2019}, month={Apr}, pages={G482–G494} }
 @article{stieler stewart_freund_blikslager_gonzalez_2018, title={Intestinal Stem Cell Isolation and Culture in a Porcine Model of Segmental Small Intestinal Ischemia}, volume={5}, ISSN={1940-087X}, url={http://dx.doi.org/10.3791/57647}, DOI={10.3791/57647}, abstractNote={Intestinal ischemia remains a major cause of morbidity and mortality in human and veterinary patients.Many disease processes result in intestinal ischemia, when the blood supply and therefore oxygen is decreased to the intestine.This leads to intestinal barrier loss and damage to the underlying tissue.Intestinal stem cells reside at the base of the crypts of Lieberkühn and are responsible for intestinal renewal during homeostasis and following injury.Ex vivo cell culture techniques have allowed for the successful study of epithelial stem cell interactions by establishing culture conditions that support the growth of three-dimensional epithelial organ-like systems (termed "enteroids" and "colonoids" from the small and large intestine, respectively).These enteroids are composed of crypt and villus-like domains and mature to contain all of the cell types found within the epithelium.Historically, murine models have been utilized to study intestinal injury.However, a porcine model offers several advantages including similarity of size as well as gastrointestinal anatomy and physiology to that of humans.By utilizing a porcine model, we establish a protocol in which segmental loops of intestinal ischemia can be created within a single animal, enabling the study of differing time points of ischemic injury and repair in vivo.Additionally, we describe a method to isolate and culture the intestinal stem cells from the ischemic loops of intestine, allowing for the continued study of epithelial repair, modulated by stem cells, ex vivo.}, number={135}, journal={Journal of Visualized Experiments}, publisher={MyJove Corporation}, author={Stieler Stewart, Amy and Freund, John M and Blikslager, Anthony T and Gonzalez, Liara M}, year={2018}, month={May} }
 @article{stewart_freund_gonzalez_2017, title={Advanced three-dimensional culture of equine intestinal epithelial stem cells}, volume={50}, ISSN={0425-1644}, url={http://dx.doi.org/10.1111/evj.12734}, DOI={10.1111/evj.12734}, abstractNote={Intestinal epithelial stem cells are critical to epithelial repair following gastrointestinal injury. The culture of intestinal stem cells has quickly become a cornerstone of a vast number of new research endeavours that range from determining tissue viability to testing drug efficacy for humans. This study aims to describe the methods of equine stem cell culture and highlights the future benefits of these techniques for the advancement of equine medicine.To describe the isolation and culture of small intestinal stem cells into three-dimensional (3D) enteroids in horses without clinical gastrointestinal abnormalities.Descriptive study.Intestinal samples were collected by sharp dissection immediately after euthanasia. Intestinal crypts containing intestinal stem cells were dissociated from the underlying tissue layers, plated in a 3D matrix and supplemented with growth factors. After several days, resultant 3D enteroids were prepared for immunofluorescent imaging and polymerase chain reaction (PCR) analysis to detect and characterise specific cell types present. Intestinal crypts were cryopreserved immediately following collection and viability assessed.Intestinal crypts were successfully cultured and matured into 3D enteroids containing a lumen and budding structures. Immunofluorescence and PCR were used to confirm the existence of stem cells and all post mitotic, mature cell types, described to exist in the horse intestinal epithelium. Previously frozen crypts were successfully cultured following a freeze-thaw cycle.Tissues were all derived from normal horses. Application of this technique for the study of specific disease was not performed at this time.The successful culture of equine intestinal crypts into 3D "mini-guts" allows for in vitro studies of the equine intestine. Additionally, these results have relevance to future development of novel therapies that harness the regenerative potential of equine intestine in horses with gastrointestinal disease (colic).}, number={2}, journal={Equine Veterinary Journal}, publisher={Wiley}, author={Stewart, A. Stieler and Freund, J. M. and Gonzalez, L. M.}, year={2017}, month={Sep}, pages={241–248} }
 @misc{stewart_pratt-phillips_gonzalez_2017, title={Alterations in Intestinal Permeability: The Role of the "Leaky Gut" in Health and Disease}, volume={52}, ISSN={["1542-7412"]}, url={https://doi.org/10.1016/j.jevs.2017.02.009}, DOI={10.1016/j.jevs.2017.02.009}, abstractNote={All species, including horses, suffer from alterations that increase intestinal permeability. These alterations, also known as “leaky gut,” may lead to severe disease as the normal intestinal barrier becomes compromised and can no longer protect against harmful luminal contents including microbial toxins and pathogens. Leaky gut results from a variety of conditions including physical stressors, decreased blood flow to the intestine, inflammatory disease, and pathogenic infections, among others. Several testing methods exist to diagnose these alterations in both a clinical and research setting. To date, most research has focused on regulation of the host immune response due to the wide variety of factors that can potentially influence the intestinal barrier. This article serves to review the normal intestinal barrier, measurement of barrier permeability, pathogenesis and main causes of altered permeability, and highlight potential alternative therapies of leaky gut in horses while relating what has been studied in other species. Conditions resulting in barrier dysfunction and leaky gut can be a major cause of decreased performance and also death in horses. A better understanding of the intestinal barrier in disease and ways to optimize the function of this barrier is vital to the long-term health and maintenance of these animals.}, journal={JOURNAL OF EQUINE VETERINARY SCIENCE}, publisher={Elsevier BV}, author={Stewart, Amy Stieler and Pratt-Phillips, Shannon and Gonzalez, Liara M.}, year={2017}, month={May}, pages={10–22} }
 @article{stewart_sanchez_mallicote_muniz_westerterp_burrow_mackay_2017, title={Effects of clarithromycin, azithromycin and rifampicin on terbutaline-induced sweating in foals}, volume={3}, DOI={10.1111/evj.12677}, abstractNote={Summary Background Erythromycin ( ERY ) induces anhidrosis in foals. Azithromycin ( AZI ) and clarithromycin ( CLA ), often combined with rifampicin ( RIF ), are commonly used to treat Rhodococcus equi infections, but effects on sweating have not been investigated. Objective To determine the effects of AZI , CLA and RIF on sweat responses in normal foals. Study design Each experiment was a blinded, duplicated, six foal × three period counterbalanced within subjects design (12 foals/experiment). Methods Antimicrobials were given orally for 5 days. In Experiment 1, ERY , AZI and CLA were given. In Experiment 2, ERY , RIF and ERY / RIF combination were used. Quantitative intradermal terbutaline sweat tests were performed daily for 3 days before and 1, 2, 5, 9, 24, and 39 days after treatment. Data were analysed by repeated measures analysis of variance procedures. Significance was P≤0.05. Results In Experiment 1, all macrolides suppressed sweating although CLA and AZI were less potent than ERY . In Experiment 2, significant sweat suppression occurred in foals given ERY with or without RIF , but there was no effect of RIF alone. Rifampicin reduced sweat suppression by ERY on Day 1 of treatment but not thereafter. Main limitations Because ERY blood concentrations were not measured, effects of RIF on ERY ‐induced anhidrosis could not definitively be ascribed to altered ERY bioavailability. Conclusions All macrolides commonly used to treat R. equi pneumonia, i.e. ERY , AZI and CLA , induce anhidrosis in foals. The potent anti‐sudorific effect of ERY is delayed, but not substantially affected by concurrent RIF administration.}, journal={Equine Veterinary Journal}, publisher={Wiley-Blackwell}, author={Stewart, A. L. Stieler and Sanchez, L. C. and Mallicote, M. F. and Muniz, A. L. and Westerterp, M. S. and Burrow, J. A. and MacKAY, R. J.}, year={2017}, month={Mar} }
 @article{taylor_mackay_nelson_stieler_roberts_castleman_2016, title={Spinal Cord Hamartomatous Myelodysplasia in 2 Horses With Clinical Neurologic Deficits}, volume={53}, DOI={10.1177/0300985815622971}, abstractNote={Two horses euthanized for neurologic deficits were diagnosed with hamartomatous myelodysplasia of the spinal cord. One was a 5-week-old Holsteiner colt exhibiting spasms of muscle rigidity in the extensor muscles of the limbs and epaxial muscles, and the other was a 3-year-old Thoroughbred colt exhibiting progressive ataxia and hypermetria in the pelvic limbs. Each had focal disorganization of the white and gray matter of the spinal cord forming a mass interspersed with neurons, glial cells, and disoriented axon bundles. In the Holsteiner colt, the mass was at the level of C 5 and included islands of meningeal tissue contiguous with the leptomeninges. The mass occluded the central canal forming hydromyelia cranial to the occlusion. In the Thoroughbred colt, the mass was at the level of L 1 on the dorsal periphery of the spinal cord and did not involve the central canal.}, number={4}, journal={Veterinary Pathology}, publisher={SAGE Publications}, author={Taylor, K. R. and MacKay, R. J. and Nelson, E. A. and Stieler, A. L. and Roberts, J. F. and Castleman, W. L.}, year={2016}, month={Jul}, pages={844–846} }
 @article{stieler_sanchez_mallicote_martabano_burrow_mackay_2015, title={Macrolide-induced hyperthermia in foals: Role of impaired sweat responses}, volume={48}, DOI={10.1111/evj.12481}, abstractNote={Summary Reasons for performing study The mechanism of hyperthermia, a potentially fatal adverse effect of erythromycin treatment of foals, is unknown. Objectives To determine the cause of erythromycin‐associated hyperthermia. It was hypothesised that the normal sweat response of foals is impaired by treatment with erythromycin. Study design Blinded, crossover study in 10 healthy pony foals. Methods Foals kept in stalls were given either erythromycin (25 mg/kg bwt orally, 3 times daily) or control for 10 days then turned out for a further 10 days. Quantitative intradermal terbutaline sweat tests were performed on Days 1 (baseline), 3, 10 and 20. The effects on terbutaline‐induced sweating of erythromycin, terbutaline concentration and treatment day were analysed by repeated‐measures ANOVA with Bonferroni‐corrected pairwise post hoc comparisons. Peak temperatures were compared by Wilcoxon's signed rank test and proportions by McNemar's related samples test. Significance was set at P<0.05. Results There were significant 2‐factor interactions for treatment × terbutaline after baseline, treatment × day at every terbutaline concentration, and day × terbutaline for erythromycin (P<0.001) but not control (P = 0.9) treatment. Sweating was significantly reduced from baseline in erythromycin‐treated foals at all subsequent days. Erythromycin‐treated foals produced less sweat at all time‐points than did control‐treated foals (P<0.05). Peak rectal temperatures of erythromycin‐treated foals were significantly higher (P = 0.02) than those of controls. During the first 3 days outside more erythromycin‐treated than control‐treated foals required treatment for hyperthermia (6 vs. 0; P = 0.03). Conclusions We believe drug‐induced anhidrosis is the likely cause of hyperthermia in some foals treated with erythromycin.}, number={5}, journal={Equine Veterinary Journal}, publisher={Wiley-Blackwell}, author={Stieler, A. L. and Sanchez, L. C. and Mallicote, M. F. and Martabano, B. B. and Burrow, J. A. and MacKay, R. J.}, year={2015}, month={Sep}, pages={590–594} }
 @article{mcnaughten_pozor_mallicote_stieler_kelleman_macpherson_2014, title={Non-Surgical management of vaginal prolapse in a late gestation alpaca (Lamos pacos)}, volume={6}, number={4}, journal={Clinical Theriogenology}, author={McNaughten, J.W. and Pozor, M.A. and Mallicote, M.F. and Stieler, A.L. and Kelleman, A.A. and MacPherson, M.L.}, year={2014}, pages={489–493} }
 @article{stieler_reuss_werpy_mackay_2013, title={What Is Your Neurologic Diagnosis?}, volume={243}, DOI={10.2460/javma.243.6.779}, abstractNote={A 2-year-old Thoroughbred filly in race training was found in its stall with signs of lethargy and periorbital swelling and a corneal ulcer in the left eye; there appeared to be no vision in that eye. These signs were attributed to unobserved head trauma. Over a 3-day period, medical management of the ulcer with topical administration of antimicrobials and atropine was unsuccessful, and the horse was referred for further evaluation. The horse had been treated with acupuncture 2 months earlier for suspected pelvic limb weakness. At the referral examination, the upper eyelid of the left eye was swollen with chemosis}, number={6}, journal={Journal of the American Veterinary Medical Association}, publisher={American Veterinary Medical Association (AVMA)}, author={Stieler, Amy L. and Reuss, Sarah M. and Werpy, Natasha M. and MacKay, Robert J.}, year={2013}, month={Sep}, pages={779–781} }
 @article{stieler_bernardo_donovan_2012, title={Neutrophil and monocyte function in neonatal dairy calves fed fresh or frozen colostrum}, volume={10}, number={4}, journal={International Journal of Applied Research in Veterinary Medicine}, author={Stieler, A.L. and Bernardo, B.S. and Donovan, G.A.}, year={2012}, pages={328–334} }