Progress 07/15/17 to 07/14/20
Outputs
Target Audience:Pork industry, veterinarians, physiologists, and nutritionists. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?
Nothing Reported
How have the results been disseminated to communities of interest?Publications. What do you plan to do during the next reporting period to accomplish the goals?
Nothing Reported
Impacts
What was accomplished under these goals?
Aim 1 We worked on assessing the role of ischemic injury as a model of hypoxic injury & circulatory re-distribution noted in heat stress on porcine mucosa. Our prior data on tight junction protein internalization has been confirmed in nursery age pigs. We assessed ischemic injury in suckling piglets. We noted a disparity in the level of injury, which related to the differing anatomy of the age groups. The elongated villi appeared to place suckling neonates at increased risk of injury compared to nursery age pigs with shorter villi. Neonates are not usually susceptible to heat stress, this data indicates over-heating in suckling pigs could be problematic with brief period of ischemia. We studied alterations of small intestine in weaned pigs during & after PEDV infection. 2 groups including 4-week-old & 8-week-old piglets were investigated. Each group contained 64 animals randomly arranged in 2 treatments [PEDV-infected (n = 40) & mock (n = 24)] & orally inoculated with PEDV IN19338 strain or mock. At day after inoculation (dpi) 2, 4, 6 & 10, pigs were euthanized. Jejunum, ileum, tonsil, spleen, & mesenteric lymph node were collected for histological examination & detection of PEDV. The 8-week-old group showed PEDV antigen was detected in PEDV-inoculated pigs in 30% (3/10) at dpi 4, 40% (4/10) at dpi 6, & 20% (2/10) at dpi 10. Compared with the mock, only PEDV-infected pigs at dpi 4 & 6 had shortened (p<0.05) villi in the jejunum. Using immunochemistry & Halo image analysis, PEDV-inoculated pigs at dpi 10 revealed lower (p<0.05) expression of Zonula occludens-1 (ZO-1) in both jejunum and ileum. ZO-1 is one tight junction proteins expressed between enterocytes & is crucial for maintaining intestinal integrity. Data suggests in weaned pigs, PEDV infection persistently reduces tight junction expression, even after apparent villus recovery, & may continue to reduce barrier function to at least dpi 10. In the 4-week-olds, PEDV antigen was detected in PEDV-inoculated pigs in 80% (8/10) at dpi 2 & 100% (10/10) at dpi 4 & 6. Compared with the mock, PEDV-infected pigs showed shortened (p<0.05) villi in the jejunum at dpi 2, 4 & 6 & ileum at dpi 4. These data imply 4-week-olds were more susceptible to PEDV infection than 8-week-old piglets. To evaluate the effect of PEDV infection in mucosal immunity, expression of polymeric Immunoglobulin Receptor (pIgR) was studied by immunochemistry & Halo image analysis. pIgR is a receptor synthesized in crypts and facilitates transcytosis of immunoglobulin A (Ig A) into enteric lumen, & IgA is the predominant antibody for mucosal immune defense. PEDV-inoculated pigs at dpi 4 had higher (p<0.05) expression of pIgR in ileum as compared with the mock. Also, pIgR expression was related to the ratio of villous length & crypt depth (lower ration, higher expression). The results suggest epithelial injury might stimulate pIgR expression in ileum during PEDV infection. Further research for evaluating impaired degrees of tight junctions between enterocytes & other changes in mucosal immune defense in PEDV-inoculated weaned pigs is undergoing. Aim 2 We continued to determine the mechanisms of tight junction re-assembly in nursery age pigs following ischemic injury. We found the basic mechanism of epithelial restitution is completely defective in neonates. This makes neonates susceptible to the effects of ischemic injury (& by interference heat stress) because they have a markedly reduced reparative response. In a highly translatable porcine model, we characterized efficient restitution of ischemia-injured intestinal epithelium to rapidly restore barrier function in juvenile (6-8 weeks of age) pigs. We found this rapid restitution is absent in younger neonatal (2-week-old) pigs, which has potentially critical relevance to higher mortality rates in younger patients with intestinal ischemic disease. Also, this neonatal repair defect can be rescued by direct application of the homogenized ischemia-injured juvenile mucosa. This homogenized mucosa includes an array of cell populations supporting complex signaling pathways known to regulate intestinal barrier homeostasis. One identified cell population that is associated with the epithelium is a network of enteric glial cells that release paracrine factors, such as pro- epidermal growth factor (EGF), critical to epithelial repair. We were interested in the glial network since it is immature at birth in rodents & may help explain this neonatal restitution defect & its rescue by the juvenile microenvironment. We hypothesized that rescue of the mucosal repair signaling in neonates is due to replacement of insufficient pro-reparative signals from an immature enteric glial network in the subepithelial compartment. We examined epithelial migration signaling & the development of the enteric glial network in neonatal and juvenile jejunal mucosa by RNAseq, western blot, immunofluorescence and iDISCO analyses. We confirmed deficiencies of known restitution signaling mediators in the neonatal mucosa as compared to juvenile mucosa, including significantly reduced EGF & annexin A2 mRNA expression as well as markedly lower protein levels of the active form of focal adhesion kinase. We found a less dense subepithelial glial network during the neonatal period & found enrichment of glial markers GFAP & S-100Beta in juvenile mucosa as compared to neonates. In vitro experiments showed the addition of juvenile pig glia-conditioned medium or pro-EGF enhanced wound restitution in IPEC-J2 cells, a neonatal pig intestinal epithelial line. These findings have provided clues as to the role of a developing glial network in the postnatal development of intestinal barrier repair mechanisms. Ongoing studies will shift into advanced mechanistic in vitro models to examine developmental glial-epithelial interactions that drive barrier restitution after ischemic injury. Identifying rescuable defects in neonatal intestinal repair mechanisms will lead to novel clinical interventions to reduce morbidity & mortality in neonatal patients suffering from intestinal ischemic injury. Aim 3 We tested the effects of dietary oligosaccharides on postnatal changes in the enteric glial cell network & intestinal morphology in a neonatal pig model, based on the finding that neonates are unable to mount a viable recovery response. We attributed this to a reduced ability to restitute, & the possibility that this is attributable to a reduced development of the enteric nervous system in neonates. We 1st addressed defects in epithelial restitution. After suckling colostrum for 24-hours, 1-day-old pigs were grouped onto 1 of 3 formula-based diets: control, high oligosaccharide (1:1 mixture of galactooligosaccharide & polydextrose, 8g/L), or low oligosaccharide (4g/L). Small intestine & colon samples were collected at 7 14 & 21 days-of-age for western blot & histological analysis. Initial histological results indicate a trend toward a decreased small intestinal villus length in the high oligosaccharide group at 7 day olds, indicative of accelerated intestinal maturity. Expression of the EGC marker glial fibrillary acidic protein is increased in the small intestinal mucosa at 7 & 14 days-of-age in the high oligosaccharide group based on preliminary western blots. Following ongoing work to assess crypt morphology & quantify additional EGC markers S100b, PLP-1, Sox10 in the small intestine & 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
- Type:
Other
Status:
Published
Year Published:
2019
Citation:
1. Ziegler AL, Pridgen T, Odle J, Van 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:
2. 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
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Progress 07/15/18 to 07/14/19
Outputs
Target Audience:Pork Industry, veterinarians, physiologists, and nutritionists. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?Training for graduate students in research, publications and presentations. How have the results been disseminated to communities of interest?Publications and presentation in Chicago, IL What do you plan to do during the next reporting period to accomplish the goals?Specific Objective 3: Determine if experimental or nutritional alteration of arachidonic acid (ARA) metabolism enhances repair in pigs subjected to heat stress. We are currently performing a nutrition trial to assess the role of oligosaccharides on maturation of the gut, including glial cells.
Impacts
What was accomplished under these goals?
Specific Objective 1: Determine the effect of heat, hypoxia, and PEDv challenges on TJ protein internalization in porcine intestinal epithelium In the 2018-2019 year, we studied alterations of small intestine in weaned pigs during and after PEDV infection. Two groups including 4-week-old and 8-week-old piglets were investigated. Each group contained sixty-four animals which were randomly arranged in two treatments [PEDV-infected (n = 40) and mock (n = 24)] and orally inoculated with PEDV IN19338 strain or mock. At day after inoculation (dpi) 2, 4, 6, and 10, pigs were euthanized, and jejunum, ileum, tonsil, spleen, and mesenteric lymph node were collected for histological examination and detection of PEDV. In the 8-week-old group, results show that PEDV antigen was detected in PEDV-inoculated pigs in 30% (3/10) at dpi 4, 40% (4/10) at dpi 6, and 20% (2/10) at dpi 10. Compared with the mock, only PEDV-infected pigs at dpi 4 and 6 had shortened (p<0.05) villi in the jejunum. However, using immunochemistry and Halo image analysis, PEDV-inoculated pigs at dpi 10 revealed lower (p<0.05) expression of Zonula occludens-1 (ZO-1) in both jejunum and ileum. ZO-1 is one of the tight junction proteins expressed between enterocytes and is crucial for maintaining intestinal integrity. These data suggest, in weaned pigs, PEDV infection persistently reduces tight junction expression, even after apparent villus recovery, and may thereby continue to reduce barrier function to at least dpi 10. In the 4-week-old group, PEDV antigen was detected in PEDV-inoculated pigs in 80% (8/10) at dpi 2 and 100% (10/10) at dpi 4 and 6. Compared with the mock, PEDV-infected pigs showed shortened (p<0.05) villi in the jejunum at dpi 2, 4, and 6 and ileum at dpi 4. These data imply 4-week-old piglets were more susceptible to PEDV infection than 8-week-old piglets. To evaluate the effect of PEDV infection in mucosal immunity, expression of polymeric Immunoglobulin Receptor (pIgR) was studied by immunochemistry and Halo image analysis. pIgR is a receptor synthesized in crypts and facilitates transcytosis of immunoglobulin A (Ig A) into enteric lumen, and IgA is the predominant antibody for mucosal immune defense. Results showed that compared with the mock, PEDV-inoculated pigs at dpi 4 had higher (p<0.05) expression of pIgR in ileum. Also, pIgR expression was related to the ratio of villous length and crypt depth; when the ratio was lower, the expression was higher. The results suggest that epithelial injury might stimulate pIgR expression in ileum during PEDV infection. Further research for evaluating impaired degrees of tight junctions between enterocytes and other changes in mucosal immune defense in PEDV-inoculated weaned pigs is undergoing. Specific Objective 2: Determine the mechanisms of TJ re-assembly following ischemia and hyperthermia in porcine intestinal epithelium We have continued to determine the mechanisms of tight junction re-assembly in nursery age pigs following ischemic injury. However, we came upon the intriguing finding that the more basic mechanism of epithelial restitution is completely defective in neonates. In a highly translatable porcine model, we have characterized efficient restitution of ischemia-injured intestinal epithelium to rapidly restore barrier function in juvenile (6-8-weeks-of-age) pigs. We recently discovered that this rapid restitution is strikingly absent in younger neonatal (2-week-old) pigs, which has potentially critical relevance to the higher mortality rates seen in younger patients with intestinal ischemic disease. We also discovered that, remarkably, this neonatal repair defect can be rescued by direct application of the homogenized ischemia-injured juvenile mucosa. This homogenized mucosa includes an array of cell populations supporting complex signaling pathways known to regulate intestinal barrier homeostasis. One recently identified cell population which is intimately associated with the epithelium is a network of enteric glial cells which release paracrine factors, such as pro- epidermal growth factor (EGF), that are critical to epithelial repair. We became particularly interested in the glial network because it is known to be immature at birth in rodents and may therefore help explain this neonatal restitution defect and its rescue by the juvenile microenvironment. Therefore, we hypothesized that rescue of the mucosal repair signaling in neonates is due to replacement of insufficient pro-reparative signals from an immature enteric glial network in the subepithelial compartment. We examined epithelial migration signaling and the development of the enteric glial network in neonatal and juvenile jejunal mucosa by RNAseq, western blot, immunofluorescence and iDISCO analyses. We confirmed deficiencies of known restitution signaling mediators in the neonatal mucosa as compared to juvenile mucosa, including significantly reduced EGF and annexin A2 mRNA expression as well as markedly lower protein levels of the active form of focal adhesion kinase (pFAK). We also discovered a less dense subepithelial glial network during the neonatal period and found enrichment of glial markers GFAP and S-100b in juvenile mucosa as compared to neonates. Finally, preliminary in vitro experiments showed that the addition of juvenile pig glia-conditioned medium or pro-EGF enhanced wound restitution in IPEC-J2 cells, a neonatal pig intestinal epithelial line. These findings have provided important clues as to the role of a developing glial network in the postnatal development of intestinal barrier repair mechanisms. Ongoing studies will shift into advanced mechanistic in vitro models to examine developmental glial-epithelial interactions which drive barrier restitution after ischemic injury. Identifying rescuable defects in neonatal intestinal repair mechanisms will lead to novel clinical interventions to reduce morbidity and mortality in neonatal patients suffering from intestinal ischemic injury.
Publications
- Type:
Journal Articles
Status:
Published
Year Published:
2018
Citation:
1. Ziegler AL, Pridgen TA, Mills JK, Gonzalez LM, Van Landeghem L, Odle J, Blikslager AT. Epithelial restitution defect in neonatal jejunum is rescued by juvenile mucosal homogenate in a pig model of intestinal ischemic injury and repair.PLoS One. 2018 Aug 23;13(8):e0200674
- Type:
Journal Articles
Status:
Published
Year Published:
2018
Citation:
2. Curry SM, Burrough ER, Schwartz KJ, Yoon KJ, Lonergan SM, Gabler NK. Porcine epidemic diarrhea virus reduces feed efficiency in nursery pigs. J Anim Sci. 2018;96:85-97
- Type:
Conference Papers and Presentations
Status:
Accepted
Year Published:
2018
Citation:
1. Ziegler AL, Pridgen TA, Gonzalez LM, Odle J, Blikslager AT. Piglets have reduced epithelial wound healing associated with an immature enteric glial cell network. Conference for Research Workers in Animal Disease, Chicago, IL, November 2018
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Progress 07/15/17 to 07/14/18
Outputs
Target Audience:Pork industry, veterinarians, physiologists, and nutritionists. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?Graduate students had the opportunity to present at the following (1) NC State research forum (2) the Digestive Disease Week (largest meeting of gastroentorologists in the world) in Washington DC, and (3) UNC Chapel Hill Center for Gastrointesitnal Biology and Disease (CGIBD) research competition. How have the results been disseminated to communities of interest?Dr. Blikslager visited the Department of Animal Science at Iowa State University. His visit included one on one conversations with potential collaborators, small group discussions to develop grant ideas, and a presentation of the current data to a university audience. What do you plan to do during the next reporting period to accomplish the goals?Continue to work on Specifice Objective 2. Start wor on Specific Objective 3: Determine if experimental or nutritional alteration of arachidonic acid (ARA) metabolism enhances repair in pigs subjected to heat stress.
Impacts
What was accomplished under these goals?
Specific Objective 1: Determine the effect of heat, hypoxia, and PEDv challenges on TJ protein internalization in porcine intestinal epithelium We have worked intensively on assessing the role of ischemic injury as a model of both hypoxic injury and the circulatory re-distribution noted in heat stress on porcine mucosa. Our prior data on tight junction protein internalization has been confirmed in nursery age pigs, but we also assessed injury in suckling piglets. Here, we noted a marked disparity in the level of injury, which related to the differing anatomy of these age groups. Initiated work on Specific Objective 2: Determine the mechanisms of TJ re-assembly following ischemia and hyperthermia in porcine intestinal epithelium
Publications
- Type:
Journal Articles
Status:
Published
Year Published:
2017
Citation:
Ziegler AL, Blikslager AT. Impaired intestinal barrier function and relapsing digestive disease: Lessons from a porcine model of early life stress. Neurogastroenterol Motil. 2017;29:1-4.
- Type:
Journal Articles
Status:
Published
Year Published:
2018
Citation:
Curry SM, Burrough ER, Schwartz KJ, Yoon KJ, Lonergan SM, Gabler NK. Porcine epidemic diarrhea virus reduces feed efficiency in nursery pigs. J Anim Sci. 2018 Feb 15;96(1):85-97
- Type:
Conference Papers and Presentations
Status:
Accepted
Year Published:
2018
Citation:
1. Ziegler AL, Pridgen TA, Gonzalez LM, Odle J, Blikslager AT. Neonates have reduced epithelial wound healing that is rescued by juvenile mucosal homogenate. CGIBD - UNC Annual Research Competition, Chapel Hill, NC, 2018
- Type:
Conference Papers and Presentations
Status:
Awaiting Publication
Year Published:
2017
Citation:
Ziegler AL, Pridgen TA, Gonzalez LM, Odle J, Blikslager AT. Neonates have reduced epithelial wound healing associated with fewer enteric glial cells as compared to juveniles in a pig model of intestinal ischemic injury and repair. FASEB: Gastrointestinal Tract XVII, Steamboat Springs, CO, 2017
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