BIOACTIVE NUTRIENTS ENHANCE INTESTINAL NEURAL NETWORKS AND RESCUE MUCOSAL REPAIR IN DEVELOPING NEONATES

• Sponsoring Institution: National Institute of Food and Agriculture
• Project Status: COMPLETE
• Funding Source: AFRI COMPETITIVE GRANT
• Reporting Frequency: Annual
• Accession No.: 1018590
• Grant No.: 2019-67017-29372
• Cumulative Award Amt.: $499,999.00
• Proposal No.: 2018-07985
• Project Start Date: Apr 1, 2019
• Project End Date: Mar 31, 2023
• Grant Year: 2019
• Program Code: [A1341]- Food Safety, Nutrition, and Health: Function and Efficacy of Nutrients

Project Director
Blikslager, A.

Recipient Organization
NORTH CAROLINA STATE UNIV
COLLEGE OF VETERINARY MEDICINE
RALEIGH,NC 27606

Performing Department
Department of Clinical Science

Non Technical Summary
Our long-term goal is to understand the interplay between nutrition and gut health using a porcine model of ischemic intestinal injury to investigate mechanisms of mucosal repair. In preliminary studies, we have shown that dietary provision of arachidonate (ARA) to neonates with ischemic intestinal injury hastens repair. Nonetheless, recovery of injured mucosa remains markedly impaired in neonates compared to juvenile animals. This led us to investigate oligosaccharides, which alter the microbiome to induce maturation of enteric neural elements involved in gut barrier function. Our central hypothesis is that intestinal repair of ischemic injury in porcine neonates can be synergistically rescued by a combination of (1) dietary oligosaccharides, that will interact with the microbiome to accelerate development of the mucosal EGC network and thus increase the bioavailability of glial-derived pro-reparative factors, and (2) ARA-derived eicosanoids which will augment closure of interepithelial tight junctions within restituting epithelium. Our specific objectives are: 1) Determine the ability of fed oligosaccharides to induce structural changes in enteric microbial communities that hasten the development of the EGC network; and 2) Determine if supplementation of oligosaccharides and ARA synergistically hastens recovery of mucosal barrier function in ischemic-injured neonatal intestine by stimulating EGC-regulated restitution and closure of associated tight junctions respectively. The proposed experiments will use novel physiologic, cellular, and immunolabeling approaches to define development and repair of porcine mucosa under the influence of two bioactive food components. We believe the proposed studies will lead to an understanding of the mechanistic connections between bioactive nutrients and gut health.

Animal Health Component

25%

Research Effort Categories

Basic

75%

Applied

25%

Developmental

0%

Classification

Knowledge Area (KA)	Subject of Investigation (SOI)	Field of Science (FOS)	Percent
305	3510	1050	50%
305	3510	1020	50%

Knowledge Area
305 - Animal Physiological Processes;

Subject Of Investigation
3510 - Swine, live animal;

Field Of Science
1050 - Developmental biology; 1020 - Physiology;

Keywords

development

enteric glial cell

gut health

repair

swine

Goals / Objectives
The central hypothesis is that intestinal repair of ischemic injury in porcine neonates can be synergistically rescued by a combination of (1) dietary oligosaccharides, which will interact with the microbiome to accelerate development of the mucosal EGC network and thus increase the bioavailability of glial-derived factors promoting restitiution, and (2) ARA-derived eicosanoids, which will augment closure of interepithelial tight junctions within restituting epithelium. To test the hypothesis, we have the following specific objectives:1. Determine the ability of fed oligosaccharides to induce structural changes in enteric microbial communities that hasten the development of the EGC network. 2. Determine if supplementation of oligosaccharides and ARA synergistically hastens recovery of mucosal barrier function in ischemic-injured neonatal intestine by stimulating EGC-regulated restitution and closure of associated tight junction respectively.

Project Methods
Anesthesia and surgery. Juvenile pigs will be sedated with xylazine/ ketamine (0.05mg/kg/11mg/kg IV respectively). For both neonates and juvenile pigs, anesthesia will be induced withisoflurane vaporized in 100% O2. Pigs will be orotracheally intubated, and maintained onisoflurane. Pigs will be placed on a water-circulated heating blanket. Pigs will have an ear veincatheterized for administration of fluids (15ml/ kg/ hr). Monitoring will include physical signs ofdepth of anesthesia, as well as pulse oximetry. The abdomen will be approached via a routinemidline incision. Jejunal or colonic loops (10cm in length) will be created by ligating the intestinallumen and mesenteric vasculature with 3-0 silk. Following 30-minutes of ischemia, pigs will beeuthanized with an overdosage of pentobarbital, after which tissues will be taken.Ussing chambers. Mucosa will be stripped from the seromuscular layer in oxygenated Ringers,and mounted in Ussing chambers with an aperture of 1.1 cm2, and bathed on the serosal andmucosal sides with porcine Ringer's solution. The serosal bathing solution will contain 10mMglucose to maintain mucosal viability. This will be osmotically balanced on the mucosal side with10mM mannitol. Bathing solutions will be oxygenated (95% O2/5% CO2) and circulated in waterjacketedreservoirs with a temperature maintained at 380C. The potential difference (PD), shortcircuit current (Isc) and TER will be continuously measured using automated voltage clamps(WPI). Mucosal-to-serosal fluxes of 3H-mannitol will be performed as described in our priorstudies.EGC primary culture. Primary cultures of porcine EGC will be obtained according to methodsdeveloped and validated in the Van Landeghem Lab. Briefly, intestines will be microdissectedand mucosa-submucosa will be isolated. Tissues will be incubated in EDTA-based buffers toremove epithelial cells, enzymatically digested and mechanically dissociated. Cultures will beenriched for EGC using selective poly-L-lysine-based coating and G5 supplement-containingmedia. Purity of EGC primary cultures will be assessed by immunofluorescence, and only EGCcultures presenting more than 80% of GFAP-, Sox10-, and S100β-positive cells will be used.Immunofluorescence, confocal and iDISCO microscopy. For confocal and traditional IFmicroscopy studies, tissues will be fixed with absolute methanol and stored in OCT at -80°C.Tissues will be thawed, rinsed in PBS, blocked with normal serum, incubated overnight at 4°C in10 antibody solutions, washed and incubated in fluorescent-tagged 20 antibodies. Tissues will bemounted in fluorescent mounting media and examined with an inverted microscope equippedwith a confocal laser scanning system. iDISCO clearing and staining will be performed usingmethods described in Renier et al59 using a Lavision Ultramicroscope II light-sheet system and theImaris analysis software at the Microscopy Services Laboratory (MSL) at UNC-CH(https://www.med.unc.edu/microscopy/).Western and cell fraction analyses. Intestinal mucosal scrapings or cell extracts will be snapfrozen and stored at -80°C before SDS-PAGE. Triton X-soluble and -insoluble fractions(representative of cytosolic and membrane cell fractions respectively) will be extracted by usingTriton X-soluble and Triton X-insoluble extraction buffers, as described in our previous studies.Tissue extracts equalized by protein concentration will be subjected to SDS-PAGE and blottingperformed using standard techniques. We will use antibodies we have validated for the pig.

Progress 04/01/19 to 03/31/23

Outputs
Target Audience:Veterinary scientists, nutritionists, animal scientists, and physiologists. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?The project has provided an advanced level of knowledge to graduate students of neonatal nutrition, and the study of development of the intestine, and the developing microbiome. More specifically, students have focused on development of the enteric nervous system, including computer-assisted whole mount imaging and western analyses. How have the results been disseminated to communities of interest?Oral presentationsat scientific Conferences(of American Physiology Summit; and of Research Workers in Animal Disease), in addition to multiple poster abstract conference presentions. What do you plan to do during the next reporting period to accomplish the goals? Nothing Reported

Impacts
What was accomplished under these goals? Specific Objective 1: Determine the ability of fed oligosaccharides to induce structural changes in enteric microbial communities that hasten the development of the EGC network After suckling colostrum for 24-hours, 40 one-day-old pigs were grouped onto one of three formula-based diets: control, high oligosaccharide (1:1 mixture of galactooligosaccharide and polydextrose, 8g/L), or low oligosaccharide (4g/L). Small intestine and colon samples were collected at 7- 14- and 21-days-of-age for TEER and mannitol flux measurement, microbiome sequencing and quantification, western blot analysis, iDISCO imaging of the EGC network and standard histology. Samples of luminal contents from the ileum and colon have sequenced revealing a definitive effect of diet on alpha diversity, and ongoing work will quantify the changes. Histological results indicate a trend toward a decreased small intestinal villus length in the high oligosaccharide group at 7-days-of-age, indicative of accelerated intestinal maturity. Expression of the EGC marker glial fibrillary acidic protein is increased in the small intestinal mucosa at 7- and 14-days-of-age in the high oligosaccharide group. There are no significant alterations of baseline barrier function as measured by TEER and flux for any group, indicating there are no detected detrimental effects on the pigs' intestinal function by oligosaccharide supplementation. iDISCO samples have been co-labeled with GFAP, S100B and Sox10 and all of the samples have been processed and imaged with the light sheet microscope. These data are undergoing complete analysis for alterations of three-dimensional glial networks induced by diet interventions using software algorithms that recently have been optimized on initial samples. Prebiotic-fed colon had lower levels of the EGC markers glial fibrillary acidic protein (GFAP) and S-100B at day 21 (P<.050) by western blot, and subjectively reduced density of GFAP+ and S-100B+ EGC were noted in preliminary analysis of iDISCO imaging of prebiotic-fed jejunal submucosa at days 14 and 21. Specific Objective 2: Determine if supplementation of oligosaccharides and ARA synergistically hastens recovery of mucosal barrier function in ischemic-injured neonatal intestine by stimulating EGC-regulated restitution and closure of associated tight junctions respectively. After suckling colostrum for 24-hours, 40 one-day-old pigs were grouped onto control or high oligosaccharide formula diets. Pigs were anesthetized at 14- days-of-age for surgical induction of 30-minutes and in a second group, 45-minutes of colonic and jejunal ischemia followed by ex vivo recovery while monitoring TEER and mannitol flux and samples were taken for standard histology. EGC were cultured from uninjured tissues of 14-day-old pigs and migration abilities, calcium responses to ATP, paracrine effects on IPEC-J2 cell restitution, and protein secretome were assessed in jejunal and colonic EGC cultures. With 30-minutes ischemia, there was no change in TEER with injury in either group indicating resistance to barrier injury in the colon. Histology indicated increased incidence of restituting epithelial cells in the injured oligosaccharide-fed small intestine where epithelial wounds were present in support of our hypothesis. Following 45-minutes ischemia, low initial TEER recovered to control levels in the control-fed colon but not prebiotic-fed colon (diet and injury interaction, P=.038), while diet had no effect on jejunal TEER recovery. Studies to better optimize the duration of colonic injury are ongoing. EGC were cultured from uninjured tissues of 14-day-old pigs fed control or prebiotic diets and migration abilities, calcium responses to ATP, paracrine effects on IPEC-J2 cell restitution, and protein secretome were assessed in jejunal and colonic EGC cultures. EGC from prebiotic-fed colonic submucosa showed decreased chemotactic motility toward sterile-filtered colonic contents (P=.010), decreased intracellular calcium response to ATP (P=.0075), and their co-culture with IPEC-J2 enhanced epithelial restitution versus monoculture (P=.032). Oppositely, EGC from prebiotic-fed jejunal submucosa showed increased intracellular calcium response (P=.050) and their co-culture with IPEC-J2 did not enhance restitution as efficiently (P=.33) as those from control-fed jejunal submucosa (P=.019). Supernatants from cultured EGC were assessed by mass spectrometry revealing several potential target proteins which are differentially secreted between the two groups. We are currently pursuing two candidate of particular interest, thymosin beta-4, which inhibits actin polymerization and is known to regulate wound healing, and Annexin A2, which is known to regulate intestinal barrier restitution. In a separate but parallel trial, we have also treated juvenile pigs with EGC inhibitor fluoroacetate and shown that this suppresses GFAP induction in EGC and inhibits epithelial repair, recapitulating the neonatal phenotype and further supporting EGC as a critical player in signaling epithelial barrier repair in developing pigs. In another study, our surgical ischemia and recovery model was further optimized by examining the effects of a period of environmental acclimation versus acute transport stress on mucosal barrier repair after intestinal ischemic injury, jejunal ischemia was induced in juvenile pigs which had been allowed to acclimate to a biomedical research housing environment or had been transported immediately prior to injury. Mucosa was recovered ex vivo while measuring transepithelial electrical resistance (TEER) and mannitol flux, and histology was assessed. Acute transport stress increases mucosal susceptibility to epithelial loss, but also primes the tissue for a more robust barrier repair response. Brief environmental acclimation, however, increases leak pathway but protects against epithelial loss during ischemia.

Publications

• Type: Conference Papers and Presentations Status: Accepted Year Published: 2023 Citation: Jodka S, Erwin SJ, Van Landeghem L, Odle J, Blikslager AT, Ziegler AL. Assessing early postnatal development of the enteric glial network. Accepted Abstract for Poster Presentation. American Physiology Summit, 20-23 April 2023. Long Beach, CA, USA.
• Type: Conference Papers and Presentations Status: Accepted Year Published: 2023 Citation: Caldwell M, Van Landeghem L, Blikslager AT, Ziegler AL. Neonatal Enteric Glia Enhance Intestinal Epithelial Restitution in vitro Following Exposure to Sterile Colonic Luminal Content of Mature but not Neonatal Pigs. Accepted Abstract for Oral Presentation. American Physiology Summit, 20-23 April 2023. Long Beach, CA, USA.
• Type: Conference Papers and Presentations Status: Published Year Published: 2022 Citation: A. L. Ziegler, C. A. Deck, M. L. Caldwell, T. A. Pridgen, E. C. Rose, A. E. Sheridan, S. J. Erwin, B. A. Wieland, A. R. Hattenhauer, C. L. Mariant, L. Van Landeghem, J. Odle, A. T. Blikslager. Dietary Oligosaccharides Differentially Modulate Microbiome, Enteric Glia, and Epithelial Barrier Function in the Neonatal Jejunum and Colon. Oral Abstract Presentation, Conference of Research Workers in Animal Disease. 20-24 January 2022. Chicago, IL, USA.
• Type: Journal Articles Status: Accepted Year Published: 2022 Citation: Boger KD, Sheridan AE, Ziegler AL, Blikslager AT. Mechanisms and modeling of wound repair in the intestinal epithelium. Tissue barriers. 2022 June 11. doi: 10.1080/21688370.2022.2087454. PubMed PMID: 35695206. PubMed Central PMCID: pending.
• Type: Journal Articles Status: Accepted Year Published: 2022 Citation: Rose EC, Blikslager AT, Ziegler AL. Porcine models of the intestinal microbiota: The translational key to understanding how gut commensals contribute to gastrointestinal disease. Front. Vet. Sci. 2022 March 25. doi: 10.3389/fvets.2022.834598; PubMed PMID: 35400098. PubMed Central PMCID: PMC8990160.
• Type: Conference Papers and Presentations Status: Published Year Published: 2022 Citation: S. J. Erwin, M.E. Clark, M.R. Aitken, J. E. Dechant, D. Hassel, A. L. Ziegler, A. T. Blikslager. Retrospective Study Evaluating Clinical Outcomes of Foals with Surgical Strangulating Lesions of the Small Intestine. AAEP Convention Proceedings. 18-22 November 2022. San Antonio, TX, USA.
• Type: Conference Papers and Presentations Status: Published Year Published: 2022 Citation: M. L. Caldwell, C. Cook, C. Mariant, M. Touvron, A. T. Blikslager, A. L. Ziegler, L. Van Landeghem. Novel Method of Primary Culture of Enteric Glial Cells from Submucosal and Myenteric Plexi of Pig Colons. Poster Abstract. 7th Annual Triangle Society for Neuroscience Meeting. 9 Sept 2022. Cary, NC, USA.
• Type: Conference Papers and Presentations Status: Published Year Published: 2022 Citation: S. J. Erwin, J. Odle, L. Van Landeghem, A. T. Blikslager, A. L. Ziegler. iDISCO Highlights Postnatal Changes in Enteric Glial Network Development in a Comparative Pig Model. Poster Abstract. CGIBD Annual Research Competition. 14 June 2022. Chapel Hill, NC, USA.
• Type: Conference Papers and Presentations Status: Published Year Published: 2022 Citation: E. A. Hellstrom, A. L. Ziegler, A. T. Blikslager. Indications of Enteric Glial IL-1 Signaling in Equine Postoperative Ileus. Poster Abstract. CGIBD Annual Research Competition. 14 June 2022. Chapel Hill, NC, USA.
• Type: Conference Papers and Presentations Status: Published Year Published: 2022 Citation: M. L. Caldwell, L. Van Landeghem, A. T. Blikslager, A. L. Ziegler. Enteric Glia Enhance Epithelial Cell Restitution In Vitro Following Exposure to Sterile Luminal Contents. Poster Abstract. CGIBD Annual Research Competition. 14 June 2022. Chapel Hill, NC, USA.

Progress 04/01/21 to 03/31/22

Outputs
Target Audience:Veterinary Scientists, Nutritionists, animal scientists, and physiologists. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?The project has provided advanced level knowledge to undergraduate and graduate students of neonatal nutrition, and the study of development of the intestine. More specifically, students have focused on development of the enteric nervous system, including computer-assisted whole mount imaging and western analyses. The developing microbiome has also been studied. How have the results been disseminated to communities of interest?The information from this project is being disseminated via target scientifica conferences, initially including the Conference of Research Workers in Animal Disease (abstract accepted and presented November 2019) What do you plan to do during the next reporting period to accomplish the goals?To continue to have studies scheduled for Objective 2

Impacts
What was accomplished under these goals? Specific Objective 1: Determine the ability of fed oligosaccharides to induce structural changes in enteric microbial communities that hasten the development of the EGC network After suckling colostrum for 24-hours, 40 one-day-old pigs were grouped onto one of three formula-based diets: control, high oligosaccharide (1:1 mixture of galactooligosaccharide and polydextrose, 8g/L), or low oligosaccharide (4g/L). Small intestine and colon samples were collected at 7- 14- and 21-days-of-age for TEER and mannitol flux measurement, microbiome sequencing and quantification, western blot analysis, iDISCO imaging of the EGC network and standard histology. Samples of luminal contents from the ileum and colon have sequenced revealing a definitive effect of diet on alpha diversity, and ongoing work will quantify the changes. Histological results indicate a trend toward a decreased small intestinal villus length in the high oligosaccharide group at 7-days-of-age, indicative of accelerated intestinal maturity. Expression of the EGC marker glial fibrillary acidic protein is increased in the small intestinal mucosa at 7- and 14-days-of-age in the high oligosaccharide group. There are no significant alterations of baseline barrier function as measured by TEER and flux for any group, indicating there are no detected detrimental effects on the pigs' intestinal function by oligosaccharide supplementation. iDISCO samples have been co-labeled with GFAP, S100B and Sox10 and all of the samples have been processed and imaged with the light sheet microscope. These data are undergoing complete analysis for alterations of three-dimensional glial networks induced by diet interventions using software algorithms that recently have been optimized on initial samples. Prebiotic-fed colon had lower levels of the EGC markers glial fibrillary acidic protein (GFAP) and S-100B at day 21 (P<.050) by western blot, and subjectively reduced density of GFAP+ and S-100B+ EGC were noted in preliminary analysis of iDISCO imaging of prebiotic-fed jejunal submucosa at days 14 and 21. program Director: Blikslager, Anthony T. Specific Objective 2: Determine if supplementation of oligosaccharides and ARA synergistically hastens recovery of mucosal barrier function in ischemic-injured neonatal intestine by stimulating EGC-regulated restitution and closure of associated tight junctions respectively. After suckling colostrum for 24-hours, 40 one-day-old pigs were grouped onto control or high oligosaccharide formula diets. Pigs were anesthetized at 14- days-of-age for surgical induction of 30-minutes and in a second group, 45-minutes of colonic and jejunal ischemia followed by ex vivo recovery while monitoring TEER and mannitol flux and samples were taken for standard histology. EGC were cultured from uninjured tissues of 14-day-old pigs and migration abilities, calcium responses to ATP, paracrine effects on IPEC-J2 cell restitution, and protein secretome were assessed in jejunal and colonic EGC cultures. With 30-minutes ischemia, there was no change in TEER with injury in either group indicating resistance to barrier injury in the colon. Histology indicated increased incidence of restituting epithelial cells in the injured oligosaccharide-fed small intestine where epithelial wounds were present in support of our hypothesis. Following 45-minutes ischemia, low initial TEER recovered to control levels in the control-fed colon but not prebiotic-fed colon (diet and injury interaction, P=.038), while diet had no effect on jejunal TEER recovery. Studies to better optimize the duration of colonic injury are ongoing. EGC were cultured from uninjured tissues of 14-day-old pigs fed control or prebiotic diets and migration abilities, calcium responses to ATP, paracrine effects on IPEC-J2 cell restitution, and protein secretome were assessed in jejunal and colonic EGC cultures. EGC from prebiotic-fed colonic submucosa showed decreased chemotactic motility toward sterile-filtered colonic contents (P=.010), decreased intracellular calcium response to ATP (P=.0075), and their co-culture with IPEC-J2 enhanced epithelial restitution versus monoculture (P=.032). Oppositely, EGC from prebiotic-fed jejunal submucosa showed increased intracellular calcium response (P=.050) and their co-culture with IPEC-J2 did not enhance restitution as efficiently (P=.33) as those from control-fed jejunal submucosa (P=.019). Supernatants from cultured EGC were assessed by mass spectrometry revealing several potential target proteins which are differentially secreted between the two groups. We are currently pursuing two candidate of particular interest, thymosin beta-4, which inhibits actin polymerization and is known to regulate wound healing, and Annexin A2, which is known to regulate intestinal barrier restitution. In a separate but parallel trial, we have also treated juvenile pigs with EGC inhibitor fluoroacetate and shown that this suppresses GFAP induction in EGC and inhibits epithelial repair, recapitulating the neonatal phenotype and further supporting EGC as a critical player in signaling epithelial barrier repair in developing pigs. In another study, our surgical ischemia and recovery model was further optimized by examining the effects of a period of environmental acclimation versus acute transport stress on mucosal barrier repair after intestinal ischemic injury, jejunal ischemia was induced in juvenile pigs which had been allowed to acclimate to a biomedical research housing environment or had been transported immediately prior to injury. Mucosa was recovered ex vivo while measuring transepithelial electrical resistance (TEER) and mannitol flux, and histology was assessed. Acute transport stress increases mucosal susceptibility to epithelial loss, but also primes the tissue for a more robust barrier repair response. Brief environmental acclimation, however, increases leak pathway but protects against epithelial loss during ischemia.

Publications

• Type: Conference Papers and Presentations Status: Published Year Published: 2022 Citation: A. L. Ziegler, C. A. Deck, M. L. Caldwell, T. A. Pridgen, E. C. Rose, A. E. Sheridan, S. J. Erwin, B. A. Wieland, A. R. Hattenhauer, C. L. Mariant, L. Van Landeghem, J. Odle, A. T. Blikslager. Dietary Oligosaccharides Differentially Modulate Microbiome, Enteric Glia, and Epithelial Barrier Function in the Neonatal Jejunum and Colon. Poster Abstract, Experimental Biology 2022. April 2-5 2022. Philadelphia, PA, USA.
• Type: Conference Papers and Presentations Status: Published Year Published: 2021 Citation: E. C. Rose, J. Odle, A. T. Blikslager, A. L. Ziegler. Colonocytes demonstrate an agedependent defect in epithelial restitution in a pig model of colonic ischemia and repair. Poster Abstract, Conference of Research Workers in Animal Disease. December 3-7 2021. Chicago, IL, USA.
• Type: Conference Papers and Presentations Status: Awaiting Publication Year Published: 2022 Citation: Rose EC, Blikslager AT, Ziegler AL. Porcine models of the intestinal microbiota: The translational key to understanding how gut commensals contribute to gastrointestinal disease. Front. Vet. Sci. Accepted 2022 Feb 28. doi: pending; PubMed PMID: pending. PubMed Central PMCID: pending
• Type: Conference Papers and Presentations Status: Published Year Published: 2021 Citation: Erwin SJ, Blikslager AT, Ziegler AL. Age-Dependent Intestinal Repair: Implications for Foals with Severe Colic. Animals. 2021 November 23. doi: 10.3390/ani11123337. PubMed PMID: 34944114; PubMed Central PMCID: PMC8697879.
• Type: Journal Articles Status: Published Year Published: 2021 Citation: Rose EC, Odle J, Blikslager AT, Ziegler AL. Probiotics, Prebiotics and Epithelial Tight Junctions: A Promising Approach to Modulate Intestinal Barrier Function. International Journal of Molecular Sciences. 2021 June 23. doi: 10.3390/ijms22136729. PMID: 34201613. PMCID: PMC8268081.
• Type: Conference Papers and Presentations Status: Published Year Published: 2020 Citation: Ziegler AL, Pridgen TA, Blikslager AT. Environmental Stressors Affect Intestinal Permeability and Repair Responses in a Pig Intestinal Ischemia Model. Tissue Barriers. 2020 Oct 1;8(4):1832421. doi: 10.1080/21688370.2020.1832421. PubMed PMID: 33100144. PMCID: PMC7714481.
• Type: Conference Papers and Presentations Status: Published Year Published: 2021 Citation: M. L. Caldwell, L. Van Landeghem, A. T. Blikslager, A. L. Ziegler. Glial cell inhibitor fluoroacetate suppresses enteric glial cell activity marker GFAP in ischemia-injured porcine small intestine. Poster Abstract. FASEB Gastrointestinal Tract XIX. 3-4 August 2021.
• Type: Conference Papers and Presentations Status: Published Year Published: 2021 Citation: A. L. Ziegler, C. A. Deck, M. L. Caldwell, T. A. Pridgen, E. C. Rose, A. E. Sheridan, S. J. Erwin, B. A. Wieland, A. R. Hattenhauer, L. Van Landeghem, J. Odle, A. T. Blikslager. Dietary Oligosaccharides Modulate Microbiome, Enteric Glia, and Epithelial Barrier Function in the Early Postnatal Period. Poster Abstract. FASEB Gastrointestinal Tract XIX. 3-4 August 2021.
• Type: Conference Papers and Presentations Status: Published Year Published: 2021 Citation: A. L. Ziegler, M. L. Caldwell, A. E. Sheridan, T. A. Pridgen, J. Odle, L. Van Landeghem, A. T. Blikslager. An age-dependent, rescuable defect in intestinal barrier repair is associated with an immature enteric glial network in a neonatal pig model of small intestinal strangulating obstruction. Oral Abstract, International Colic Symposium, 4-7 program Director: Blikslager, Anthony T.September 2021
• Type: Conference Papers and Presentations Status: Published Year Published: 2021 Citation: . S.J. Erwin, J. Odle, L. Van Landeghem, A.T. Blikslager, A.L. Ziegler. iDISCO Highlights Postnatal Changes in Enteric Glial Network Development in a Comparative Pig Model. Poster Abstract, Gastronauts Global, 11-13 May 2021
• Type: Conference Papers and Presentations Status: Published Year Published: 2020 Citation: A.L. Ziegler, T.A. Pridgen, E.C. Rose, A.E. Sheridan, B.A. Wieland, A.R. Hattenhauer, L.C. Van Landeghem, J. Odle1, A.T. Blikslager. Dietary oligosaccharides modulate microbiome, enteric glia, and epithelial barrier function in a neonatal pig model. Oral Abstract, Conference of Research Workers in Animal Disease. 5-8 Dec 2020. Virtual.
• Type: Conference Papers and Presentations Status: Published Year Published: 2020 Citation: A.L. Ziegler, A.E. Sheridan, T.A. Pridgen, J. Odle, L.Van Landeghem, A.T. Blikslager. An age-dependent, rescuable defect in intestinal barrier repair is associated with an immature enteric glial network in a neonatal pig model of intestinal ischemia. Poster Abstract, Translational Science 2020, 14-17 April 2020. Washington, DC, USA.
• Type: Conference Papers and Presentations Status: Published Year Published: 2020 Citation: A.L. Ziegler, A.T. Blikslager. Effects of Environmental Acclimation versus Transport Stress on Barrier Recovery in a Pig Model of Intestinal Ischemia and Repair. Poster Abstract, Experimental Biology. 4-7 April 2020. San Diego, CA, USA.
• Type: Conference Papers and Presentations Status: Published Year Published: 2020 Citation: L Shapiro, AL Ziegler, J Odle, L Van Landeghem, AT Blikslager. Effects of Oligosaccharide Supplementation on Intestinal Morphology and Enteric Glial Cell Marker Expression in a Neonatal Pig Model. Poster Abstract, Experimental biology. 4-7 April 2020. San Diego, CA, USA.
• Type: Conference Papers and Presentations Status: Published Year Published: 2020 Citation: SE Erwin, M Touvron, J Odle, L Van Landeghem, AT Blikslager, AL Ziegler. iDISCO allows complete visualization and analysis of postnatal enteric nervous system development in a comparative pig model. Poster Abstract, Experimental Biology. 4-7 April 2020. San Diego, CA, USA.
• Type: Conference Papers and Presentations Status: Published Year Published: 2020 Citation: AE Sheridan, TA Pridgen, J Odle, L Van Landeghem, AT Blikslager, AL Ziegler. A Glial Cell Inhibitor Blocks Epithelial Barrier Repair in a Pig Model of Intestinal Ischemia. Poster Abstract, Experimental Biology. 4-7 April 2020. San Diego, CA, USA.
• Type: Conference Papers and Presentations Status: Published Year Published: 2020 Citation: Ziegler AL, Blikslager AT. Effects of Environmental Acclimation versus Transport Stress on Barrier Recovery in a Pig Model of Intestinal Ischemia and Repair. Accepted abstract presentation, Experimental Biology. April 4-7 2020. San Diego, CA, USA.
• Type: Conference Papers and Presentations Status: Published Year Published: 2020 Citation: Shapiro L, Ziegler AL, Odle J, Van Landeghem L, Blikslager AT. Effects of Oligosaccharide Supplementation on Intestinal Morphology and Enteric Glial Cell Marker Expression in a Neonatal Pig Model. Accepted abstract presentation, Experimental Biology. April 4-7 2020. San Diego, CA, USA
• Type: Conference Papers and Presentations Status: Published Year Published: 2020 Citation: Erwin SE, Touvron M, Odle J, Van Landeghem L, Blikslager AT, Ziegler AL. iDISCO allows complete visualization and analysis of postnatal enteric nervous system development in a comparative pig model. Accepted abstract presentation, Experimental Biology. April 4-7 2020. San Diego, CA, USA
• Type: Conference Papers and Presentations Status: Published Year Published: 2020 Citation: Sheridan AE, Pridgen TA, Odle J, Van Landeghem L, Blikslager AT, Ziegler AL. A Glial Cell Inhibitor Blocks Epithelial Barrier Repair in a Pig Model of Intestinal Ischemia. Accepted abstract presentation, Experimental Biology. April 4-7 2020. San Diego, CA, USA
• Type: Conference Papers and Presentations Status: Published Year Published: 2020 Citation: Ziegler AL, Sheridan AE, Pridgen TA, Odle J, Van Landeghem L, Blikslager AT. An age-dependent, rescuable defect in intestinal barrier repair is associated with an immature enteric glial network in a neonatal pig model of intestinal schemia. Accepted abstract presentation, Translational Science 2020. April 4-7 2020. San Diego, CA, USA

Progress 04/01/20 to 03/31/21

Outputs
Target Audience:Nutritionists, animal scientists, veterinary scientists, physiologists.? Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?The project has provided advanced level knowledge to undergraduate and graduate students of neonatal nutrition, and the study of development of the intestine. More specifically, students have focused on development of the enteric nervous system, including computer-assisted whole mount imaging and western analyses. The developing microbiome has also been studied. How have the results been disseminated to communities of interest?The information from this project is being disseminated via target scientifica conferences, initially including the Conference of Research Workers in Animal Disease (abstract accepted and presented November 2019) and Experimental Biology (abstract published, not presented because of cancellation May 2020) What do you plan to do during the next reporting period to accomplish the goals?Studies will be scheduled for Specific Objective 2: Determine if supplementation of oligosaccharides and ARA synergistically hastens recovery of mucosal barrier function in ischemic-injured neonatal intestine by stimulating EGC-regulated restitution and closure of associated tight junctions respectively.

Impacts
What was accomplished under these goals? Specific Objective 1: Determine the ability of fed oligosaccharides to induce structural changes in enteric microbial communities that hasten the development of the EGC network. We tested the effects of dietary oligosaccharides on postnatal changes in the microbiota, EGC network and intestinal morphology in a neonatal pig model. After suckling colostrum for 24-hours, one-day-old pigs were grouped into one of three formula-based diets: control, high oligosaccharide (1:1 mixture of galactooligosaccharide and polydextrose, 8g/L), or low oligosaccharide (4g/L). Small intestine and colon samples were collected at 7- 14- and 21-days-of-age for western blot and histological analysis, and fecal swabs were sequenced for 16S rDNA. Microbial taxa changed in a time- and diet-dependent manner with diets containing oligosaccharides clustering by day 7 and becoming progressively more tightly clustered over time (P<.050). TEER and histological appearance of the epithelium in non-injured jejunum and colon were unaffected by diet. Preliminary histological results indicate a trend toward a decreased small intestinal villus length in the high oligosaccharide group at 7-days-of-age, indicative of accelerated intestinal maturity. Expression of the EGC marker glial fibrillary acidic protein is increased in the small intestinal mucosa at 7- and 14-days-of-age in the high oligosaccharide group based on preliminary western blots. However, preliminary analysis of volume imaging also revealed subjectively reduced GFAP and S100b signal in the jejunal submucosa at 14- and 21-days in the oligosaccharide-fed pigs. Following ongoing work to assess crypt morphology and quantify additional EGC markers S100β, PLP-1, Sox10 in the small intestine and colon, we expect to find increased mucosal expression of EGC markers earlier in postnatal development in the high oligosaccharide group, as well as histological changes consistent with enhanced rates of gut maturation in pigs fed a high oligosaccharide diet. Understanding how dietary inputs drive intestinal development postnatally may improve practices for managing optimal gut health early in life. Specific Objective 2: Determine if supplementation of oligosaccharides and ARA synergistically hastens recovery of mucosal barrier function in ischemic-injured neonatal intestine by stimulating EGC-regulated restitution and closure of associated tight junctions respectively We hypothesized that dietary oligosaccharide supplementation accelerates postnatal microbial colonization and EGC network maturation, thus enhancing restitution after intestinal ischemic injury in neonates. Therefore, we tested the effects of prebiotic fiber supplementation on epithelial barrier recovery following ischemic injury and on EGC activity in vitro. After 24-hours of colostrum, piglets were fed control or oligosaccharide-supplemented formula for 21-days. Intestinal samples were collected at 1, 7, 14 and 21 days for analysis by western blot, three-dimensional volume imaging, and histology. Thirty-minutes of jejunal and colonic ischemic injury was induced surgically in 14-day-old pigs. Injured mucosa was recovered ex vivowhile monitoring epithelial barrier function by transepithelial electrical resistance (TEER). Colonic EGC were isolated to assess migration and calcium responses, co-culture effects on scratch-wounded IPEC-J2 cells, and to identify secreted proteins by mass spectrometry. In the injured colon, prebiotic-fed pigs demonstrated higher initial TEER (P=.0012) and injured jejunum showed increased restitution after recovery (P=.0020). The colon of prebiotic-fed pigs had lower levels of the EGC markers glial fibrillary acidic protein (GFAP) and S100b at 21-days (P<0.050). EGC from prebiotic-fed pigs showed decreased chemotactic motility toward sterile-filtered colonic contents (P≤.010) and decreased intracellular calcium response to ATP (P=.0075). In addition, prebiotic EGC differentially secreted 13 proteins of interest versus control-fed pigs. EGC from prebiotic-fed pigs enhanced IPEC-J2 restitution as compared to IPEC-J2 in monoculture (P=.032), while EGC from control-fed pigs did not. Preliminary results indicate dietary oligosaccharides exert important effects on EGC network development and activity, and on epithelial restitution in vivo and in vitro. Ongoing work will test the effects of oligosaccharides on recovery from an increased duration of ischemic injury, explore prebiotic-driven changes in jejunal EGC in vitro including targeted study of select probiotic-EGC-secreted proteins of interest. Understanding nutrient-microbiome-EGC-epithelial interactions during postnatal development may lead to novel preventive and clinical practices to improve intestinal health in vulnerable neonates.

Publications

• Type: Conference Papers and Presentations Status: Accepted Year Published: 2021 Citation: A. L. Ziegler, M. L. Caldwell, A. E. Sheridan, T. A. Pridgen, J. Odle, L. Van Landeghem, A. T. Blikslager. An age-dependent, rescuable defect in intestinal barrier repair is associated with an immature enteric glial network in a neonatal pig model of small intestinal strangulating obstruction. Oral Abstract, International Colic Symposium, 4-7 September 2021.
• Type: Conference Papers and Presentations Status: Accepted Year Published: 2021 Citation: S. J. Erwin, J. E. Dechant, D. Hassel, A. L. Ziegler, A. T. Blikslager. Multi-institutional retrospective case control study evaluating clinical outcomes of neonatal equine patients undergoing surgical correction of strangulating lesions of the small intestine: 2000-2019. Oral Abstract, International Colic Symposium, 4-7 September 2021.
• Type: Conference Papers and Presentations Status: Published Year Published: 2020 Citation: A.L. Ziegler, T.A. Pridgen, E.C. Rose, A.E. Sheridan, B.A. Wieland, A.R. Hattenhauer, L.C. Van Landeghem, J. Odle1, A.T. Blikslager. Dietary oligosaccharides modulate microbiome, enteric glia, and epithelial barrier function in a neonatal pig model. Oral Abstract, Conference of Research Workers in Animal Disease. 5-8 Dec 2020.
• Type: Conference Papers and Presentations Status: Published Year Published: 2020 Citation: S. J. Erwin, J. E. Dechant, M. R. Aitken, D. M. Hassel, A. L. Ziegler, A. T. Blikslager. Multi-institutional retrospective case-control study evaluating clinical outcomes of foals with small intestinal strangulating obstruction: 2000-2019. Oral Abstract, 2020 American College of Veterinary Surgeons Surgery E-Summit. 22-24 October 2020.
• Type: Conference Papers and Presentations Status: Published Year Published: 2020 Citation: A.L. Ziegler, A.E. Sheridan, T.A. Pridgen, J. Odle, L.Van Landeghem, A.T. Blikslager. An age-dependent, rescuable defect in intestinal barrier repair is associated with an immature enteric glial network in a neonatal pig model of intestinal ischemia. Poster Abstract, Translational Science 2020, 14-17 April 2020. Washington, DC, USA.
• Type: Conference Papers and Presentations Status: Published Year Published: 2021 Citation: S.J. Erwin, J. Odle, L. Van Landeghem, A.T. Blikslager, A.L.Ziegler. iDISCO Highlights Postnatal Changes in Enteric Glial Network Development in a Comparative Pig Model. Poster Abstract, Gastronauts Global, 11-13 May 2021.
• Type: Conference Papers and Presentations Status: Published Year Published: 2021 Citation: A. Kandakatla, O. Martinez-Uribe, N. Karlovich, M. Harbrecht , E. Hellstrom, S. Singh , A. L. Ziegler, T. Becker, V. Varadan , A. T. Blikslager, K. S. Garman. The effect of cyclooxygenase (COX)-2 on esophageal submucosal gland (ESMG) acinar ductal metaplasia. Poster Abstract, Digestive Disease Week 2021, 21-23 May 2021.
• Type: Conference Papers and Presentations Status: Published Year Published: 2020 Citation: A.L. Ziegler, A.T. Blikslager. Effects of Environmental Acclimation versus Transport Stress on Barrier Recovery in a Pig Model of Intestinal Ischemia and Repair. Poster Abstract, Experimental Biology. 4-7 April 2020. San Diego, CA, USA.
• Type: Conference Papers and Presentations Status: Published Year Published: 2020 Citation: L Shapiro, AL Ziegler, J Odle, L Van Landeghem, AT Blikslager. Effects of Oligosaccharide Supplementation on Intestinal Morphology and Enteric Glial Cell Marker Expression in a Neonatal Pig Model. Poster Abstract, Experimental Biology. 4-7 April 2020. San Diego, CA, USA.
• Type: Conference Papers and Presentations Status: Published Year Published: 2020 Citation: SE Erwin, M Touvron, J Odle, L Van Landeghem, AT Blikslager, AL Ziegler. iDISCO allows complete visualization and analysis of postnatal enteric nervous system development in a comparative pig model. Poster Abstract, Experimental Biology. 4-7 April 2020. San Diego, CA, USA.
• Type: Conference Papers and Presentations Status: Published Year Published: 2020 Citation: AE Sheridan, TA Pridgen, J Odle, L Van Landeghem, AT Blikslager, AL Ziegler. A Glial Cell Inhibitor Blocks Epithelial Barrier Repair in a Pig Model of Intestinal Ischemia. Poster Abstract, Experimental Biology. 4-7 April 2020. San Diego, CA, USA.
• Type: Conference Papers and Presentations Status: Published Year Published: 2019 Citation: Ziegler AL, Pridgen T, Odle J, Van Landeghem L, Magness S, Blikslager AT. Rescue of restitution defect in a neonatal pig intestinal ischemia model is associated with a developing enteric glial network. Gastroenterology 2019;156:S709
• Type: Conference Papers and Presentations Status: Published Year Published: 2019 Citation: Ziegler AL, Pridgen TA, Sheridan A, Odle J, Magness ST, Van Landeghem L, Blikslager AT. Suckling piglets have a rescuable defect in intestinal barrier repair associated with an immature glial cell network. Conference for research Workers in Animal Disease, Chicago, November 2019
• Type: Journal Articles Status: Published Year Published: 2021 Citation: Rose EC, Odle J, Blikslager AT, Ziegler AL. Probiotics, Prebiotics and Epithelial Tight Junctions: A Promising Approach to Modulate Intestinal Barrier Function. International Journal of Molecular Sciences. 2021 June 23. doi: 10.3390/ijms22136729. PMID: 34201613. PMCID: PMC8268081
• Type: Journal Articles Status: Published Year Published: 2020 Citation: Ziegler AL, Pridgen TA, Blikslager AT. Environmental Stressors Affect Intestinal Permeability and Repair Responses in a Pig Intestinal Ischemia Model. Tissue Barriers. 2020 Oct 1;8(4):1832421. doi: 10.1080/21688370.2020.1832421. PubMed PMID: 33100144. PMCID: PMC7714481.

Progress 04/01/19 to 03/31/20

Outputs
Target Audience:Nutritionists, animal scientists, veterinary scientists, physiologists. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?The project has provided advanced level knowledge to undergraduate and graduate students of neonatal nutrition, and the studyof development of the intestine. More specifically, students have focused on development of the enteric nervous system, includingcomputer-assisted whole mount imaging and western analyses. The developing microbiome has also been studied. How have the results been disseminated to communities of interest?The information from this project is being disseminated via target scientifica conferences, initially including the Conference of Research Workers in Animal Disease (abstract accepted and presented November 2019) and Experimental Biology (abstract published, not presented because of cancellation May 2020) What do you plan to do during the next reporting period to accomplish the goals?Studies will be scheduled for Specific Objective 2: Determine if supplementation of oligosaccharides and ARA synergistically hastens recovery of mucosal barrier function in ischemic-injured neonatal intestine by stimulating EGC-regulated restitution and closure of associated tight junctions respectively.

Impacts
What was accomplished under these goals? Specific Objective 1: Determine the ability of fed oligosaccharides to induce structural changes in enteric microbial communities that hasten the development of the EGC network. We tested the effects of dietary oligosaccharides on postnatal changes in the EGC network and intestinal morphology in a neonatal pig model. After suckling colostrum for 24-hours, one-day-old pigs were grouped onto one of three formula-based diets: control, high oligosaccharide (1:1 mixture of galactooligosaccharide and polydextrose, 8g/L), or low oligosaccharide (4g/L). Small intestine and colon samples were collected at 7- 14- and 21-days-of-age for western blot and histological analysis. Preliminary histological results indicate a trend toward a decreased small intestinal villus length in the high oligosaccharide group at 7-days-of-age, indicative of accelerated intestinal maturity. Expression of the EGC marker glial fibrillary acidic protein is increased in the small intestinal mucosa at 7- and 14-days-of-age in the high oligosaccharide group based on preliminary western blots. Following ongoing work to assess crypt morphology and quantify additional EGC markers S100b, PLP-1, Sox10 in the small intestine and colon, we expect to find increased mucosal expression of EGC markers earlier in postnatal development in the high oligosaccharide group, as well as histological changes consistent with enhanced rates of gut maturation in pigs fed a high oligosaccharide diet. Understanding how dietary inputs drive intestinal development postnatally may improve practices for managing optimal gut heath early in life.

Publications