@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} }
 @misc{ludwig_hobbs_mckinney-aguirre_gonzalez_2023, title={Biomarkers of Intestinal Injury in Colic}, volume={13}, ISSN={["2076-2615"]}, url={https://doi.org/10.3390/ani13020227}, DOI={10.3390/ani13020227}, abstractNote={Biomarkers are typically proteins, enzymes, or other molecular changes that are elevated or decreased in body fluids during the course of inflammation or disease. Biomarkers pose an extremely attractive tool for establishing diagnoses and prognoses of equine gastrointestinal colic, one of the most prevalent causes of morbidity and mortality in horses. This topic has received increasing attention because early diagnosis of some forms of severe colic, such as intestinal ischemia, would create opportunities for rapid interventions that would likely improve case outcomes. This review explores biomarkers currently used in equine medicine for colic, including acute phase proteins, proinflammatory cytokines, markers of endotoxemia, and tissue injury metabolites. To date, no single biomarker has been identified that is perfectly sensitive and specific for intestinal ischemia; however, L-lactate has been proven to be a very functional and highly utilized diagnostic tool. However, further exploration of other biomarkers discussed in this review may provide the key to accelerated identification, intervention, and better outcomes for horses suffering from severe colic.}, number={2}, journal={ANIMALS}, author={Ludwig, Elsa K. and Hobbs, Kallie J. and McKinney-Aguirre, Caroline A. and Gonzalez, Liara M.}, year={2023}, month={Jan} }
 @article{veerasammy_gonzalez_baez-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={2}, ISSN={["2042-3306"]}, url={https://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.}, journal={EQUINE VETERINARY JOURNAL}, author={Veerasammy, Brittany and Gonzalez, Gabriel and Baez-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} }
 @article{ludwig_abraham_mckinney_freund_stewart_garman_barbas_sudan_gonzalez_2023, title={Comparison of the Effects of Normothermic Machine Perfusion and Cold Storage Preservation on Porcine Intestinal Allograft Regenerative Potential and Viability}, volume={107}, ISSN={["1534-6080"]}, 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}, 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={Jul}, pages={25–25} }
 @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={Abstract Background 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. Results 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. Conclusions 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{tse_meganck_araba_yount_shaffer_hou_munt_adams_wykoff_morowitz_et al._2022, title={Genomewide CRISPR knockout screen identified PLAC8 as an essential factor for SADS-CoVs infection}, volume={119}, ISSN={["1091-6490"]}, DOI={10.1073/pnas.2118126119}, abstractNote={Significance A potential outbreak of swine acute diarrhea syndrome coronavirus (SADS-CoV) in the human population could be devastating. Using genomewide CRISPR knockout screening, we identified the placenta-associated 8 protein (PLAC8) as an essential host factor for SADS-CoV infection, uncovering a novel antiviral target for CoV infection. The PLAC8-related pathway may also have implications for other CoV infections. Given the ability of SADS-CoV to infect human primary cultures without adaptation, our findings lay the foundation for pandemic preparedness for the potential emergence of SADS-CoVs in response to the One Health Initiative.}, number={18}, journal={PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA}, author={Tse, Longping V and Meganck, Rita M. and Araba, Kenza C. and Yount, Boyd L. and Shaffer, Kendall M. and Hou, Yixuan J. and Munt, Jennifer E. and Adams, Lily E. and Wykoff, Jason A. and Morowitz, Jeremy M. and et al.}, year={2022}, month={May} }
 @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"]}, DOI={10.1097/TXD.0000000000001390}, abstractNote={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.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.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.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}, 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={Nov} }
 @misc{schaaf_gonzalez_2022, title={Use of Translational, Genetically Modified Porcine Models to Ultimately Improve Intestinal Disease Treatment}, volume={9}, ISSN={["2297-1769"]}, DOI={10.3389/fvets.2022.878952}, abstractNote={For both human and veterinary patients, non-infectious intestinal disease is a major cause of morbidity and mortality. To improve treatment of intestinal disease, large animal models are increasingly recognized as critical tools to translate the basic science discoveries made in rodent models into clinical application. Large animal intestinal models, particularly porcine, more closely resemble human anatomy, physiology, and disease pathogenesis; these features make them critical to the pre-clinical study of intestinal disease treatments. Previously, large animal model use has been somewhat precluded by the lack of genetically altered large animals to mechanistically investigate non-infectious intestinal diseases such as colorectal cancer, cystic fibrosis, and ischemia-reperfusion injury. However, recent advances and increased availability of gene editing technologies has led to both novel use of large animal models in clinically relevant intestinal disease research and improved testing of potential therapeutics for these diseases.}, journal={FRONTIERS IN VETERINARY SCIENCE}, author={Schaaf, Cecilia R. and Gonzalez, Liara M.}, year={2022}, month={May} }
 @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: 1) active ISC [aISC; highly proliferative; leucine-rich-repeat-containing G protein-coupled receptor 5 (LGR5+)-positive or sex-determining region Y-box 9 -antigen Ki67-positive (SOX9+Ki67+)] and 2) reserve ISC [rISC; less proliferative; homeodomain-only protein X positive (HOPX+)]. The contributions of these ISCs have been evaluated both in vivo and in vitro using a porcine model of mesenteric vascular occlusion to understand mechanisms that modulate ISC recovery responses following ischemic injury. In our previously published work, we observed that rISC conversion to an activated state was associated with decreased HOPX expression during in vitro recovery. In the present study, we wanted to evaluate the direct role of HOPX on cellular proliferation during recovery after injury. Our data demonstrated that during early in vivo recovery, injury-resistant HOPX+ cells maintain quiescence. Subsequent early regeneration within the intestinal crypt occurs around 2 days after injury, a period in which HOPX expression decreased. When HOPX was silenced in vitro, cellular proliferation of injured cells was promoted during recovery. This suggests that HOPX may serve a functional role in ISC-mediated regeneration after injury and could be a target to control ISC proliferation.NEW & NOTEWORTHY This paper supports that rISCs are resistant to ischemic injury and likely an important source of cellular renewal following near-complete epithelial loss. Furthermore, we have evidence that HOPX controls ISC activity state and may be a critical signaling pathway during ISC-mediated repair. Finally, we use multiple novel methods to evaluate ISCs in a translationally relevant large animal model of severe intestinal injury and provide evidence for the potential role of rISCs as therapeutic targets.}, 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{vermeire_gonzalez_jansens_cox_devriendt_2021, title={Porcine small intestinal organoids as a model to explore ETEC-host interactions in the gut}, volume={52}, ISSN={["1297-9716"]}, DOI={10.1186/s13567-021-00961-7}, abstractNote={Abstract Small intestinal organoids, or enteroids, represent a valuable model to study host–pathogen interactions at the intestinal epithelial surface. Much research has been done on murine and human enteroids, however only a handful studies evaluated the development of enteroids in other species. Porcine enteroid cultures have been described, but little is known about their functional responses to specific pathogens or their associated virulence factors. Here, we report that porcine enteroids respond in a similar manner as in vivo gut tissues to enterotoxins derived from enterotoxigenic Escherichia coli , an enteric pathogen causing postweaning diarrhoea in piglets. Upon enterotoxin stimulation, these enteroids not only display a dysregulated electrolyte and water balance as shown by their swelling, but also secrete inflammation markers. Porcine enteroids grown as a 2D-monolayer supported the adhesion of an F4 + ETEC strain. Hence, these enteroids closely mimic in vivo intestinal epithelial responses to gut pathogens and are a promising model to study host–pathogen interactions in the pig gut. Insights obtained with this model might accelerate the design of veterinary therapeutics aimed at improving gut health.}, number={1}, journal={VETERINARY RESEARCH}, author={Vermeire, Bjarne and Gonzalez, Liara M. and Jansens, Robert J. J. and Cox, Eric and Devriendt, Bert}, year={2021}, month={Jun} }
 @article{vermeire_gonzalez_jansens_cox_devriendt_2021, title={Porcine small intestinal organoids as a model to explore ETEC-host interactions in the gut (vol 52, 94, 2021)}, volume={52}, ISSN={["1297-9716"]}, DOI={10.1186/s13567-021-00977-z}, number={1}, journal={VETERINARY RESEARCH}, author={Vermeire, Bjarne and Gonzalez, Liara M. and Jansens, Robert J. J. and Cox, Eric and Devriendt, Bert}, year={2021}, month={Aug} }
 @article{singh_hung_shanahan_kanke_bonfini_dame_biraud_peck_oyesola_freund_et al._2020, title={Enteroendocrine Progenitor Cell-Enriched mir-7 Regulates Intestinal Epithelial Proliferation in an Xiap-Dependent Manner}, volume={9}, ISSN={["2352-345X"]}, DOI={10.1016/j.jcmgh.2019.11.001}, abstractNote={The enteroendocrine cell (EEC) lineage is important for intestinal homeostasis. It was recently shown that EEC progenitors contribute to intestinal epithelial growth and renewal, but the underlying mechanisms remain poorly understood. MicroRNAs are under-explored along the entire EEC lineage trajectory, and comparatively little is known about their contributions to intestinal homeostasis.We leverage unbiased sequencing and eight different mouse models and sorting methods to identify microRNAs enriched along the EEC lineage trajectory. We further characterize the functional role of EEC progenitor-enriched miRNA, miR-7, by in vivo dietary study as well as ex vivo enteroid in mice.First, we demonstrate that miR-7 is highly enriched across the entire EEC lineage trajectory and is the most enriched miRNA in EEC progenitors relative to Lgr5+ intestinal stem cells. Next, we show in vivo that in EEC progenitors miR-7 is dramatically suppressed under dietary conditions that favor crypt division and suppress EEC abundance. We then demonstrate by functional assays in mouse enteroids that miR-7 exerts robust control of growth, as determined by budding (proxy for crypt division), EdU and PH3 staining, and likely regulates EEC abundance also. Finally, we show by single-cell RNA sequencing analysis that miR-7 regulates Xiap in progenitor/stem cells and we demonstrate in enteroids that the effects of miR-7 on mouse enteroid growth depend in part on Xiap and Egfr signaling.This study demonstrates for the first time that EEC progenitor cell-enriched miR-7 is altered by dietary perturbations and that it regulates growth in enteroids via intact Xiap and Egfr signaling.}, number={3}, journal={CELLULAR AND MOLECULAR GASTROENTEROLOGY AND HEPATOLOGY}, author={Singh, Ajeet P. and Hung, Yu-Han and Shanahan, Michael T. and Kanke, Matt and Bonfini, Alessandro and Dame, Michael K. and Biraud, Mandy and Peck, Bailey C. E. and Oyesola, Oyebola O. and Freund, John M. and et al.}, year={2020}, pages={447–464} }
 @article{orr_baker_lynch_hughes_clark_slone_fogle_gonzalez_2020, title={Prognostic value of colonic and peripheral venous lactate measurements in horses with large colon volvulus}, volume={1}, url={https://doi.org/10.1111/vsu.13361}, DOI={10.1111/vsu.13361}, abstractNote={To determine the prognostic value of (1) colonic venous lactate or peripheral lactate values obtained before and after manual correction of a large colon volvulus and (2) a combination of variables including pelvic flexure biopsy.Prospective clinical study.Forty adult horses in which large colon volvulus was diagnosed intraoperatively.Colonic venous, peripheral venous, and arterial blood samples were collected to measure lactate values before and after manual correction. Mucosal biopsy samples were obtained in cases that underwent enterotomy or colonic resection and anastomosis. Interstitium to crypt (I:C) ratio and hemorrhage scores were measured. Optimal cutoff values were determined by receiver operator curve analysis, and associations between variables and short-term outcome were determined by univariable regression. Short-term survival was defined as horses being discharged from the hospital. P ≤ .05 was considered significant.No association was found between colonic venous lactate values before (P = .011) or after (P = .201) manual correction of large colon volvulus and determination of short-term outcome. Peripheral venous lactate at admission ≥3.2 mmol/L and after manual correction ≥5 mmol/L, arterial lactate postmanual correction ≥3.53 mmol/L, and histomorphometric measurements of mucosal hemorrhage ≥3 and I:C ratio > 1 were associated with poor short-term outcome.Peripheral lactate values, histomorphometric measures of I:C ratio, and hemorrhage score provided prognostic information that could help guide recommendations made to owners.Peripheral lactate values after manual correction provide important intraoperative diagnostic information to assist in predicting case outcome in the operative and immediately postoperative period.}, journal={Veterinary Surgery}, publisher={Wiley}, author={Orr, Kindra. E. and Baker, W. True and Lynch, Tim M. and Hughes, Faith E. and Clark, Carol K. and Slone, Donnie E., Jr and Fogle, Callie A. and Gonzalez, Liara M.}, year={2020}, month={Apr} }
 @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{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{blikslager_gonzalez_2018, title={Equine intestinal mucosal pathobiology}, volume={6}, journal={Annual review of animal biosciences, vol 6}, author={Blikslager, A. and Gonzalez, L.}, year={2018}, pages={157–175} }
 @article{fletcher_mansell_martin_borst_barnes_gonzalez_2018, title={Gross Morphometry, Histomorphometry, and Immunohistochemistry Confirm Early and Persistent Jejunal Crypt Hyperplasia in Poults with Enteritis and Depressed Growth}, volume={62}, ISSN={0005-2086 1938-4351}, url={http://dx.doi.org/10.1637/11759-101717-reg.1}, DOI={10.1637/11759-101717-reg.1}, abstractNote={Phosphorylated histone 3 (PH3) and cleaved caspase 3 (CCASP3) were used to detect proliferating and apoptotic cells, respectively, in the jejunums of female sibling poults, with and without enteritis and depressed growth, from hatch to day 35. Poults that developed enteritis and depressed growth (SIB flock) were raised on a commercial farm in eastern North Carolina, whereas poults with normal growth and no enteritis (TAU flock) were raised in the Teaching Animal Unit at North Carolina State University College of Veterinary Medicine. Beginning on day 5 through day 35 and at processing, TAU poults were significantly heavier than SIB poults. Jejunal weights, relative jejunal weights, and jejunal densities were greater in SIB poults from day 10 through 35. Jejunal efficiency (body weight /jejunal length) was higher in TAU poults at day 5 and days 10 through 35. Mucosal thickness was greater in SIB poults between days 7 and 21 but greater in TAU poults at days 28 and 35. From day 7 to 35, villus-to-crypt ratios were higher for TAU poults and lower for SIB poults because hyperplastic crypts formed a greater percentage of the mucosa in SIB poults. By day 7, PH3- and CCASP3-positive cells were increased in SIB poults, showing that mucosal changes resulted from combined crypt epithelial hyperplasia and increased apoptosis of villous enterocytes. Findings in this study confirm that enteritis, in the absence of clinical signs, and depressed growth in turkey poults begins by day 7, can be identified microscopically, persists for at least 35 days, is associated with lower processing weights, and has a profound negative effect on turkey growth.}, number={2}, journal={Avian Diseases}, publisher={American Association of Avian Pathologists (AAAP)}, author={Fletcher, O. J. and Mansell, R. and Martin, M. P. and Borst, L. B. and Barnes, H. John and Gonzalez, L. M.}, year={2018}, month={Jun}, pages={163–170} }
 @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{gonzalez_2018, title={Large colon volvulus: The continued conundrum}, volume={30}, ISSN={["2042-3292"]}, url={https://doi.org/10.1111/eve.12748}, DOI={10.1111/eve.12748}, number={6}, journal={EQUINE VETERINARY EDUCATION}, publisher={Wiley}, author={Gonzalez, L. M.}, year={2018}, month={Jun}, pages={312–314} }
 @article{royal_hunt_gonzalez_lewbart_bailey_2018, title={Veterinary Medical Students' Motivations for Exercise}, volume={45}, ISSN={0748-321X 1943-7218}, url={http://dx.doi.org/10.3138/jvme.0117-004r}, DOI={10.3138/jvme.0117-004r}, abstractNote={The Centers for Disease Control (CDC) declares exercise to be one of the most important activities one can do to improve health. The benefits of exercise are well documented and include both physiologic and psychological health. Given the current landscape of wellness issues in veterinary medical education, it is necessary that students engage in exercise activities to manage stress and increase overall health. Therefore, to develop targeted interventions with the greatest likelihood for success, it is first necessary to understand what motivates veterinary medical students to exercise given their unique situational and environmental factors. This study is the first to explore this issue systematically in veterinary medical education, thus it is the authors' hope that the findings from this research will help identify exercise-related wellness interventions that could be implemented in veterinary medical schools.}, number={3}, journal={Journal of Veterinary Medical Education}, publisher={University of Toronto Press Inc. (UTPress)}, author={Royal, Kenneth D. and Hunt, Suzanne A. and Gonzalez, Liara M. and Lewbart, Gregory A. and Bailey, Kate M.}, year={2018}, month={Aug}, pages={367–373} }
 @article{royal_hunt_gonzalez_lewbart_bailey_2018, title={Veterinary Medical Students' Motivations for Exercise}, volume={1}, ISSN={0748-321X 1943-7218}, url={http://dx.doi.org/10.3138/jvme.0117-004}, DOI={10.3138/jvme.0117-004}, abstractNote={The Centers for Disease Control (CDC) declares exercise to be one of the most important activities one can do to improve health. The benefits of exercise are well documented and include both physiologic and psychological health. Given the current landscape of wellness issues in veterinary medical education, it is necessary that students engage in exercise activities to manage stress and increase overall health. Therefore, to develop targeted interventions with the greatest likelihood for success, it is first necessary to understand what motivates veterinary medical students to exercise given their unique situational and environmental factors. This study is the first to explore this issue systematically in veterinary medical education, thus it is the authors' hope that the findings from this research will help identify exercise-related wellness interventions that could be implemented in veterinary medical schools.}, journal={Journal of Veterinary Medical Education}, publisher={University of Toronto Press Inc. (UTPress)}, author={Royal, Kenneth D. and Hunt, Suzanne A. and Gonzalez, Liara M. and Lewbart, Gregory A. and Bailey, Kate M.}, year={2018}, month={Jan}, pages={1–7} }
 @article{dellon_joshi_blikslager_gonzalez_pridgen_whitlow_ivanovic_slaughter_garman_karp_et al._2017, title={A Novel Inflammation-Activated Drug Delivery System Using Self-Assembling Hydrogel Doubles Esophageal Dwell Time in an Esophageal Injury Porcine Model}, volume={152}, ISSN={0016-5085}, url={http://dx.doi.org/10.1016/S0016-5085(17)32956-6}, DOI={10.1016/S0016-5085(17)32956-6}, number={5}, journal={Gastroenterology}, publisher={Elsevier BV}, author={Dellon, Evan S. and Joshi, Nitin and Blikslager, Anthony and Gonzalez, Liara M. and Pridgen, Tiffany and Whitlow, Ann and Ivanovic, Marija and Slaughter, Kai and Garman, Katherine S. and Karp, Jeffrey M. and et al.}, year={2017}, month={Apr}, pages={S859–S860} }
 @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={Summary Background 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. Objectives To describe the isolation and culture of small intestinal stem cells into three‐dimensional (3D) enteroids in horses without clinical gastrointestinal abnormalities. Study design Descriptive study. Methods 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. Results 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. Main limitations Tissues were all derived from normal horses. Application of this technique for the study of specific disease was not performed at this time. Conclusions 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 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{kruger_gonzalez_pridgen_mccall_furstenberg_harnden_carnighan_cox_blikslager_garman_et al._2017, title={Ductular and proliferative response of esophageal submucosal glands in a porcine model of esophageal injury and repair}, volume={313}, ISSN={0193-1857 1522-1547}, url={http://dx.doi.org/10.1152/ajpgi.00036.2017}, DOI={10.1152/ajpgi.00036.2017}, abstractNote={Esophageal injury is a risk factor for diseases such as Barrett’s esophagus (BE) and esophageal adenocarcinoma. To improve understanding of signaling pathways associated with both normal and abnormal repair, animal models are needed. Traditional rodent models of esophageal repair are limited by the absence of esophageal submucosal glands (ESMGs), which are present in the human esophagus. Previously, we identified acinar ductal metaplasia in human ESMGs in association with both esophageal injury and cancer. In addition, the SOX9 transcription factor has been associated with generation of columnar epithelium and the pathogenesis of BE and is present in ESMGs. To test our hypothesis that ESMGs activate after esophageal injury with an increase in proliferation, generation of a ductal phenotype, and expression of SOX9, we developed a porcine model of esophageal injury and repair using radiofrequency ablation (RFA). The porcine esophagus contains ESMGs, and RFA produces a consistent and reproducible mucosal injury in the esophagus. Here we present a temporal assessment of this model of esophageal repair. Porcine esophagus was evaluated at 0, 6, 18, 24, 48, and 72 h and 5 and 7 days following RFA and compared with control uninjured esophagus. Following RFA, ESMGs demonstrated an increase in ductal phenotype, echoing our prior studies in humans. Proliferation increased in both squamous epithelium and ESMGs postinjury with a prominent population of SOX9-positive cells in ESMGs postinjury. This model promises to be useful in future experiments evaluating mechanisms of esophageal repair. NEW & NOTEWORTHY A novel porcine model of injury and repair using radiofrequency ablation has been developed, allowing for reproducible injury to the esophagus to study repair in an animal model with esophageal submucosal glands, a key anatomical feature and missing in rodent models but possibly harboring progenitor cells. There is a strong translational component to this porcine model given the anatomical and physiological similarities between pigs and humans.}, number={3}, journal={American Journal of Physiology-Gastrointestinal and Liver Physiology}, publisher={American Physiological Society}, author={Kruger, L. and Gonzalez, Liara and Pridgen, T. A. and McCall, S. J. and Furstenberg, R. J. and Harnden, I. and Carnighan, G. E. and Cox, A. M. and Blikslager, Anthony and Garman, K. S. and et al.}, year={2017}, month={Sep}, pages={G180–G191} }
 @article{von furstenberg_li_stolarchuk_feder_campbell_kruger_gonzalez_blikslager_cardona_mccall_et al._2017, title={Porcine Esophageal Submucosal Gland Culture Model Shows Capacity for Proliferation and Differentiation}, volume={4}, ISSN={2352-345X}, url={http://dx.doi.org/10.1016/j.jcmgh.2017.07.005}, DOI={10.1016/j.jcmgh.2017.07.005}, abstractNote={Although cells comprising esophageal submucosal glands (ESMGs) represent a potential progenitor cell niche, new models are needed to understand their capacity to proliferate and differentiate. By histologic appearance, ESMGs have been associated with both overlying normal squamous epithelium and columnar epithelium. Our aim was to assess ESMG proliferation and differentiation in a 3-dimensional culture model.We evaluated proliferation in human ESMGs from normal and diseased tissue by proliferating cell nuclear antigen immunohistochemistry. Next, we compared 5-ethynyl-2'-deoxyuridine labeling in porcine ESMGs in vivo before and after esophageal injury with a novel in vitro porcine organoid ESMG model. Microarray analysis of ESMGs in culture was compared with squamous epithelium and fresh ESMGs.Marked proliferation was observed in human ESMGs of diseased tissue. This activated ESMG state was recapitulated after esophageal injury in an in vivo porcine model, ESMGs assumed a ductal appearance with increased proliferation compared with control. Isolated and cultured porcine ESMGs produced buds with actively cycling cells and passaged to form epidermal growth factor-dependent spheroids. These spheroids were highly proliferative and were passaged multiple times. Two phenotypes of spheroids were identified: solid squamous (P63+) and hollow/ductal (cytokeratin 7+). Microarray analysis showed spheroids to be distinct from parent ESMGs and enriched for columnar transcripts.Our results suggest that the activated ESMG state, seen in both human disease and our porcine model, may provide a source of cells to repopulate damaged epithelium in a normal manner (squamous) or abnormally (columnar epithelium). This culture model will allow the evaluation of factors that drive ESMGs in the regeneration of injured epithelium. The raw microarray data have been uploaded to the National Center for Biotechnology Information Gene Expression Omnibus (accession number: GSE100543).}, number={3}, journal={Cellular and Molecular Gastroenterology and Hepatology}, publisher={Elsevier BV}, author={von Furstenberg, Richard J. and Li, Joy and Stolarchuk, Christina and Feder, Rachel and Campbell, Alexa and Kruger, Leandi and Gonzalez, Liara M. and Blikslager, Anthony T. and Cardona, Diana M. and McCall, Shannon J. and et al.}, year={2017}, month={Nov}, pages={385–404} }
 @article{kucera_stranahan_hughes_blikslager_gonzalez_2017, title={Protein biomarker of cell proliferation determines survival to discharge in cases of equine large colon volvulus}, volume={50}, ISSN={0425-1644}, url={http://dx.doi.org/10.1111/evj.12767}, DOI={10.1111/evj.12767}, abstractNote={Progenitor cells play critical roles in epithelial repair following ischaemic injury. Protein biomarkers have been used to identify intestinal progenitor cell subpopulations. This study aims to determine if a critical number of intestinal progenitor cells can predict tissue viability and survival to discharge of large colon volvulus (LCV) cases.The objectives were to 1) identify intestinal progenitor cell subpopulations using biomarkers: proliferating cell nuclear antigen (PCNA), sex determining region Y box 9 (SOX9), phospho-histone H3 (PHH3) and Ki-67, 2) define cut-off values for critical numbers of positive cells and 3) determine if survival to discharge is associated with cut-off values.Retrospective cohort study.Adult horses admitted to the Farm and Equine Veterinary Medical Center at NC State's Veterinary Hospital and Peterson and Smith Equine Hospital between 2006 and 2016 that underwent an exploratory coeliotomy with a diagnosis of LCV of ≥360 degrees, had pelvic flexure biopsy and that recovered from general anaesthesia were selected for inclusion in the study. Immunohistochemical analyses were performed and positive cells were counted. Optimal cut-off values were determined using receiver operator curves. A Fisher's exact test was used to associate cut-off values with survival to discharge.In this study, 23 cases of LCV ≥360° were included. Of 23 horses, 13 (57%) survived to discharge. A cut-off value of <2.1 PHH3 positive cells per crypt correctly predicted death with 100% sensitivity (95% CI; 69.15-100%) and 84.62% specificity (95% CI; 54.55-98.08%). LCV cases with <2.1 PHH3 positive cells per crypt were 96.6 times more likely to die (95% CI; 4.14-2255 and P < 0.0001). Biomarkers PCNA, SOX9 and Ki-67 did not predict short-term survival.The population size was small.PHH3 immunohistochemical analysis may assist in more accurate prediction of survival to hospital discharge of LCV cases. The summary is available in Spanish - see Supporting Information.}, number={4}, journal={Equine Veterinary Journal}, publisher={Wiley}, author={Kucera, C. R. and Stranahan, L. W. and Hughes, F. and Blikslager, A. T. and Gonzalez, L. M.}, year={2017}, month={Nov}, pages={452–456} }
 @article{lima_lin_jacobi_man_sommer_flowers_blikslager_gonzalez_odle_2017, title={Supplementation of Maternal Diets with Docosahexaenoic Acid and Methylating Vitamins Impacts Growth and Development of Fetuses from Malnourished Gilts}, volume={2}, ISSN={2475-2991}, url={http://dx.doi.org/10.3945/cdn.117.001958}, DOI={10.3945/cdn.117.001958}, abstractNote={Like many species, pregnant swine mobilize and repartition body nutrient stores during extreme malnutrition to support fetal development.The objective of this study was to model chronic human maternal malnutrition and measure effects of methylating-vitamins (MVs, containing choline, folate, B-6, B-12, and riboflavin) and docosahexaenoic acid (DHA) supplementation on fetal growth and development.Pregnant gilts (n = 24) were either fully nourished (2.0 kg/d) with a corn-plus-isolated-soy-protein basal diet (control) supplemented with MVs and DHA or nourishment was restricted throughout gestation. Basal diet fed to malnourished gilts was reduced progressively from 50% to 70% restriction (1.0 to 0.6 kg/d) and was supplemented following a 2 (±MVs) x 2 (±DHA) factorial design. Full-term c-sections were performed to assess impacts on low and normal birth weight (LBW/NBW) fetuses (n = 238).Body weight gain of malnourished gilts was 10% of full-fed control dams (P < 0.05), but offspring birth weight, length, girth, and percentage of LBW fetuses were not different between treatments. The number of pigs per litter was reduced by 30% in malnourished control dams. Fetal brain weights were reduced by 7% compared to positive controls (P < 0.05). Micronutrient supplementation to malnourished dams increased fetal brain weights back to full-fed control levels. Dams with DHA produced offspring with higher DHA concentrations in brain and liver (P < 0.05). Plasma choline concentration was 4-fold higher in fetuses from unsupplemented malnourished dams (P < 0.0001). Global DNA methylation status of fetuses from restricted dams was higher than in control fetuses, including brain, liver, heart, muscle, and placenta tissues (P < 0.05). Addition of DHA increased methylation in LBW fetal brains (P < 0.05).Despite the mobilization of maternal stores, malnourished litters displayed reduced brain development that was fully mitigated by micronutrient supplementation. Severe maternal malnutrition increased global DNA methylation in several fetal tissues that was unaltered by choline and B-vitamin supplementation.}, number={3}, journal={Current Developments in Nutrition}, publisher={Oxford University Press (OUP)}, author={Lima, Hope K and Lin, Xi and Jacobi, Sheila K and Man, Caolai and Sommer, Jeffrey and Flowers, William and Blikslager, Anthony and Gonzalez, Liara and Odle, Jack}, year={2017}, month={Dec} }
 @article{simpson_gonzalez_chung_blikslager_magness_piedrahita_2016, title={27 AN IMPROVED LARGE ANIMAL MODEL FOR THE STUDY OF ADULT STEM CELLS}, volume={28}, ISSN={1031-3613}, url={http://dx.doi.org/10.1071/RDV28N2AB27}, DOI={10.1071/RDV28N2AB27}, abstractNote={Murine models for the study of adult stem cell populations have broadened the understanding of previously uncharacterized stem cell niches. The development of murine reporter lines for the leucine-rich repeat-containing G-protein-coupled receptor-5 (Lgr5) has highlighted the importance of this gene as a stem cell marker in the stomach, intestine, hair follicle, liver, and kidney in mice. These models however have significant limitations in terms of translational applications because of anatomical and physiological differences between humans and mice. In order to overcome these limitations, we have sought to develop a porcine LGR5 reporter line. We report the generation of a porcine stem cell reporter line using the combination of transcription activator-like effector nucleases and somatic cell NT. Transcription activator-like effector nuclease-mediated homologous recombination was used to drive the integration of an internal ribosome entry site green fluorescent protein fusion into the 3′ untranslated region of the LGR5 locus in porcine fetal fibroblast cells. Multiple cell lines were developed and screened for the proper integration event. Upon confirmation of proper integration by genomic DNA sequencing, these lines were used as donors for somatic cell NT. Transfer of the somatic cell NT reconstructed embryos to a surrogate gilt resulted in 3 live births, and the establishment of a founder line of LGR5-green fluorescent protein reporter pigs. We have begun to characterise these lines, having observed fluorescent labelling of putative stem cell populations in the intestinal crypts and hair follicles from these animals. Many of these observations parallel the expression patterns observed in similar murine models. We have confirmed the fluorescent reporter signal by immunohistochemistry using an anti-green fluorescent protein antibody, and are working towards colocalization studies using anti-LGR5 antibodies and RNA in situ hybridization, as well as the characterisation of additional stem cell populations in the pig. The development of this line of transgenic pigs represents significant progress toward the study of adult stem cells, their progenitors, and the stem cell niche, using a large animal model with an anatomy, physiology, and ability to recapitulate human disease that overcomes the current limitations of rodent models. Funding was provided by NIH R21OD019738.}, number={2}, journal={Reproduction, Fertility and Development}, publisher={CSIRO Publishing}, author={Simpson, S. and Gonzalez, L. and Chung, J. and Blikslager, A. and Magness, S. and Piedrahita, J.}, year={2016}, pages={143} }
 @article{royal_gonzalez_2016, title={An Evaluation of the Psychometric Properties of an Advising Survey for Medical and Professional Program Students}, volume={6}, ISSN={1927-0534 1927-0526}, url={http://dx.doi.org/10.5539/jedp.v6n1p195}, DOI={10.5539/jedp.v6n1p195}, abstractNote={<p>The purpose of this study was to evaluate the psychometric properties of a newly developed instrument intended to measure faculty competence as it pertains to their role as advisors, particularly in medical and professional programs. A total of 166 students completed the Faculty Advisor’s Skills and Behaviors Inventory (FASBI). The psychometric properties of the FASBI were evaluated using the Rasch Rating Scale Model. Results indicate the FASBI is a psychometrically-sound instrument capable of producing valid and reproducible measures.</p>}, number={1}, journal={Journal of Educational and Developmental Psychology}, publisher={Canadian Center of Science and Education}, author={Royal, Kenneth D. and Gonzalez, Liara M.}, year={2016}, month={Apr}, pages={195} }
 @inbook{gonzalez_2016, place={Hoboken, NJ}, edition={3rd}, title={Intestinal Stem Cells}, booktitle={Equine Acute Abdomen}, publisher={Wiley & Sons, Inc}, author={Gonzalez, L.M.}, editor={Blikslager, A.T. and White, N.A. and Moore, J.N. and Mair, T.S.Editors}, year={2016} }
 @inbook{gonzalez_2016, place={Hoboken, NJ}, edition={3rd}, title={Intestinal Viability}, booktitle={Equine Acute Abdomen}, publisher={Wiley & Sons, Inc}, author={Gonzalez, L.M.}, editor={Blikslager, A.T. and White, N.A. and Moore, J.N. and Mair, T.S.Editors}, year={2016}, pages={570} }
 @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} }
 @article{kruger_gonzalez_von furstenberg_henning_blikslager_garman_2016, title={Mo1253 Ductular and Proliferative Response of Esophageal Submucosal Glands in a Porcine Model of Esophageal Injury and Repair}, volume={150}, ISSN={0016-5085}, url={http://dx.doi.org/10.1016/S0016-5085(16)32312-5}, DOI={10.1016/S0016-5085(16)32312-5}, number={4}, journal={Gastroenterology}, publisher={Elsevier BV}, author={Kruger, Leandi and Gonzalez, Liara M. and von Furstenberg, Richard J. and Henning, Susan J. and Blikslager, Anthony and Garman, Katherine S.}, year={2016}, month={Apr}, pages={S679–S680} }
 @article{stewart_freund_magness_lund_blikslager_gonzalez_2016, title={Mo1298 Critical Contribution of Intestinal Stem Cells in the Repair of Ischemia Reperfusion Injury}, volume={150}, ISSN={0016-5085}, url={http://dx.doi.org/10.1016/S0016-5085(16)32355-1}, DOI={10.1016/S0016-5085(16)32355-1}, number={4}, journal={Gastroenterology}, publisher={Elsevier BV}, author={Stewart, Amy S. and Freund, John and Magness, Scott and Lund, Pauline K. and Blikslager, Anthony and Gonzalez, Liara M.}, year={2016}, month={Apr}, pages={S691} }
 @article{gonzalez_moeser_blikslager_2015, title={Animal models of ischemia-reperfusion-induced intestinal injury: progress and promise for translational research}, volume={308}, ISSN={0193-1857 1522-1547}, url={http://dx.doi.org/10.1152/ajpgi.00112.2013}, DOI={10.1152/ajpgi.00112.2013}, abstractNote={Research in the field of ischemia-reperfusion injury continues to be plagued by the inability to translate research findings to clinically useful therapies. This may in part relate to the complexity of disease processes that result in intestinal ischemia but may also result from inappropriate research model selection. Research animal models have been integral to the study of ischemia-reperfusion-induced intestinal injury. However, the clinical conditions that compromise intestinal blood flow in clinical patients ranges widely from primary intestinal disease to processes secondary to distant organ failure and generalized systemic disease. Thus models that closely resemble human pathology in clinical conditions as disparate as volvulus, shock, and necrotizing enterocolitis are likely to give the greatest opportunity to understand mechanisms of ischemia that may ultimately translate to patient care. Furthermore, conditions that result in varying levels of ischemia may be further complicated by the reperfusion of blood to tissues that, in some cases, further exacerbates injury. This review assesses animal models of ischemia-reperfusion injury as well as the knowledge that has been derived from each to aid selection of appropriate research models. In addition, a discussion of the future of intestinal ischemia-reperfusion research is provided to place some context on the areas likely to provide the greatest benefit from continued research of ischemia-reperfusion injury.}, number={2}, journal={American Journal of Physiology-Gastrointestinal and Liver Physiology}, publisher={American Physiological Society}, author={Gonzalez, Liara M. and Moeser, Adam J. and Blikslager, Anthony T.}, year={2015}, month={Jan}, pages={G63–G75} }
 @article{gonzalez_kinnin_blikslager_2015, title={Characterization of discrete equine intestinal epithelial cell lineages}, volume={76}, ISSN={0002-9645}, url={http://dx.doi.org/10.2460/ajvr.76.4.358}, DOI={10.2460/ajvr.76.4.358}, abstractNote={Abstract OBJECTIVE To characterize epithelial cells of the small intestine and colon in horses without clinical gastrointestinal abnormalities with an emphasis on the stem cell niche constituents. SAMPLE Mucosal biopsy specimens from small and large intestines obtained from 12 horses euthanized for reasons unrelated to gastrointestinal disease or systemic disease. PROCEDURES Intestinal biopsy specimens were collected by sharp dissection immediately following euthanasia. Specimens were prepared for immunohistochemical, immunofluorescence, and transmission electron microscopic imaging to detect and characterize each epithelial cell type. Antibodies against protein biomarkers for cellular identification were selected on the basis of expression in other mammalian species. RESULTS Intestinal epithelial cell types were identified by means of immunostaining and morphological characterization with transmission electron microscopy. Some differences in biomarker expression and antibody cross-reactivity were identified in equine tissue, compared with other species. However, each known type of mucosal epithelial cell was identified in equine tissue. CONCLUSIONS AND CLINICAL RELEVANCE The methodology used can enhance detection of stem cells and progenitor cells as well as postmitotic cell lineages in equine intestinal tissues. Results may have relevance to regenerative potential of intestinal mucosa and survival in horses with colic.}, number={4}, journal={American Journal of Veterinary Research}, publisher={American Veterinary Medical Association (AVMA)}, author={Gonzalez, Liara M. and Kinnin, Leslie A. and Blikslager, Anthony T.}, year={2015}, month={Apr}, pages={358–366} }
 @article{gonzalez_moeser_blikslager_2015, title={Porcine models of digestive disease: the future of large animal translational research}, volume={166}, ISSN={1931-5244}, url={http://dx.doi.org/10.1016/j.trsl.2015.01.004}, DOI={10.1016/j.trsl.2015.01.004}, abstractNote={There is increasing interest in nonrodent translational models for the study of human disease. The pig, in particular, serves as a useful animal model for the study of pathophysiological conditions relevant to the human intestine. This review assesses currently used porcine models of gastrointestinal physiology and disease and provides a rationale for the use of these models for future translational studies. The pig has proven its utility for the study of fundamental disease conditions such as ischemia-reperfusion injury, stress-induced intestinal dysfunction, and short bowel syndrome. Pigs have also shown great promise for the study of intestinal barrier function, surgical tissue manipulation and intervention, as well as biomaterial implantation and tissue transplantation. Advantages of pig models highlighted by these studies include the physiological similarity to human intestine and mechanisms of human disease. Emerging future directions for porcine models of human disease include the fields of transgenics and stem cell biology, with exciting implications for regenerative medicine.}, number={1}, journal={Translational Research}, publisher={Elsevier BV}, author={Gonzalez, Liara M. and Moeser, Adam J. and Blikslager, Anthony T.}, year={2015}, month={Jul}, pages={12–27} }
 @article{gonzalez_2015, title={The mother of a gut cell: Intestinal epithelial stem cells}, volume={27}, ISSN={["2042-3292"]}, DOI={10.1111/eve.12456}, abstractNote={Equine Veterinary EducationVolume 27, Issue 11 p. 559-560 Editorial The mother of a gut cell: Intestinal epithelial stem cells L. M. Gonzalez, L. M. Gonzalez Clinical Sciences, North Carolina State University, Raleigh, USASearch for more papers by this author L. M. Gonzalez, L. M. Gonzalez Clinical Sciences, North Carolina State University, Raleigh, USASearch for more papers by this author First published: 13 October 2015 https://doi.org/10.1111/eve.12456Citations: 4Read the full textAboutPDF ToolsExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinked InRedditWechat No abstract is available for this article.Citing Literature Volume27, Issue11November 2015Pages 559-560 RelatedInformation}, number={11}, journal={EQUINE VETERINARY EDUCATION}, author={Gonzalez, L. M.}, year={2015}, month={Nov}, pages={559–560} }
 @article{gonzalez_magness_lund_blikslager_2014, title={Mo1960 Intestinal Epithelial Cells Expressing Biomarkers of Crypt Base Columnar or Reserve Stem Cells Show Differential Resistance to Ischemia-Reperfusion Injury}, volume={146}, ISSN={0016-5085}, url={http://dx.doi.org/10.1016/S0016-5085(14)62550-6}, DOI={10.1016/S0016-5085(14)62550-6}, number={5}, journal={Gastroenterology}, publisher={Elsevier BV}, author={Gonzalez, Liara M. and Magness, Scott T. and Lund, Pauline K. and Blikslager, Anthony}, year={2014}, month={May}, pages={S-702} }
 @article{gonzalez_fogle_baker_hughes_law_motsinger-reif_blikslager_2014, title={Operative factors associated with short-term outcome in horses with large colon volvulus: 47 cases from 2006 to 2013}, volume={47}, ISSN={0425-1644}, url={http://dx.doi.org/10.1111/evj.12273}, DOI={10.1111/evj.12273}, abstractNote={There is an important need for objective parameters that accurately predict the outcome of horses with large colon volvulus.To evaluate the predictive value of a series of histomorphometric parameters on short-term outcome, as well as the impact of colonic resection on horses with large colon volvulus.Retrospective cohort study.Adult horses admitted to the Equine and Farm Animal Veterinary Center at North Carolina State University, Peterson and Smith and Chino Valley Equine Hospitals between 2006 and 2013 that underwent an exploratory coeliotomy, diagnosed with large colon volvulus of ≥360 degrees, where a pelvic flexure biopsy was obtained, and that recovered from general anaesthesia, were selected for inclusion in the study. Logistic regression was used to determine associations between signalment, histomorphometric measurements of interstitium-to-crypt ratio, degree of haemorrhage, percentage loss of luminal and glandular epithelium, as well as colonic resection with short-term outcome (discharge from the hospital).Pelvic flexure biopsies from 47 horses with large colon volvulus were evaluated. Factors that were significantly associated with short-term outcome on univariate logistic regression were Thoroughbred breed (P = 0.04), interstitium-to-crypt ratio >1 (P = 0.02) and haemorrhage score ≥3 (P = 0.005). Resection (P = 0.92) was not found to be associated significantly with short-term outcome. No combined factors increased the likelihood of death in forward stepwise logistic regression modelling. A digitally quantified measurement of haemorrhage area strengthened the association of haemorrhage with nonsurvival in cases of large colon volvulus.Histomorphometric measurements of interstitium-to-crypt ratio and degree of haemorrhage predict short-term outcome in cases of large colon volvulus. Resection was not associated with short-term outcome in horses selected for this study. Accurate quantification of mucosal haemorrhage at the time of surgery may improve veterinary surgeons' prognostic capabilities in horses with large colon volvulus.}, number={3}, journal={Equine Veterinary Journal}, publisher={Wiley}, author={Gonzalez, L. M. and Fogle, C. A. and Baker, W. T. and Hughes, F. E. and Law, J. M. and Motsinger-Reif, A. A. and Blikslager, A. T.}, year={2014}, month={May}, pages={279–284} }
 @article{gonzalez_williamson_piedrahita_blikslager_magness_2013, title={Cell lineage identification and stem cell culture in a porcine model for the study of intestinal epithelial regeneration}, volume={8}, number={6}, journal={PLoS One}, author={Gonzalez, L. M. and Williamson, I. and Piedrahita, J. A. and Blikslager, A. T. and Magness, S. T.}, year={2013} }
 @article{gonzalez_blikslager_piedrahita_magness_2013, title={Su1099 A Translational Porcine Model of Intestinal Stem Cells}, volume={144}, ISSN={0016-5085}, url={http://dx.doi.org/10.1016/S0016-5085(13)61466-3}, DOI={10.1016/S0016-5085(13)61466-3}, number={5}, journal={Gastroenterology}, publisher={Elsevier BV}, author={Gonzalez, Liara and Blikslager, Anthony and Piedrahita, Jorge A. and Magness, Scott T.}, year={2013}, month={May}, pages={S-398} }
 @article{gonzalez_schramme_robertson_thrall_redding_2010, title={MRI FEATURES OF METACARPO(TARSO)PHALANGEAL REGION LAMENESS IN 40 HORSES}, volume={51}, ISSN={1058-8183 1740-8261}, url={http://dx.doi.org/10.1111/j.1740-8261.2010.01676.x}, DOI={10.1111/j.1740-8261.2010.01676.x}, abstractNote={Lameness originating from the metacarpo(tarso)phalangeal (MP) joint has a significant effect on the use and athletic competitiveness of a horse. The identification of the cause of lameness originating from the MP joint can be challenging, given the limitations of radiography, ultrasonography, and nuclear scintigraphy. Our purpose was to describe the injury types and incidence in magnetic resonance imaging (MRI) studies from 40 horses with lameness attributable to the MP joint region where it was not possible to reach a clinically plausible diagnosis using other imaging modalities. Horses were examined in a 1.5 T magnet (Siemens Medical Solutions) under general anesthesia. The frequency of occurrence of MR lesions was subchondral bone injury (19), straight or oblique distal sesamoidean desmitis (13), articular cartilage injury and osteoarthritis (eight), suspensory branch desmitis (seven), osteochondral fragmentation (seven), proximal sesamoid bone injury (seven), inter-sesamoidean desmitis (four), deep digital flexor tendonitis (four), collateral desmitis (three), superficial digital flexor tendonitis (two), enostosis-like lesions of the proximal phalanx or MCIII (two), desmitis of the palmar annular ligament (one), desmitis of the proximal digital annular ligament (one), and dystrophic calcification of the lateral digital extensor tendon (one). Twenty-five horses had multiple MR abnormalities. MRI provided information that was complementary to radiography, ultrasonography, and nuclear scintigraphy and that allowed for a comprehensive evaluation of all structures in the MP joint region and a diagnosis in all 40 horses.}, number={4}, journal={Veterinary Radiology & Ultrasound}, publisher={Wiley}, author={Gonzalez, Liara M. and Schramme, Michael C. and Robertson, Ian D. and Thrall, Donald E. and Redding, Rich W.}, year={2010}, month={Apr}, pages={404–414} }