Digestive Diseases and Sciences

, Volume 61, Issue 6, pp 1524–1533 | Cite as

Tight Junction Ultrastructure Alterations in a Mouse Model of Enteral Nutrient Deprivation

  • Farokh R. DemehriEmail author
  • Susanne M. Krug
  • Yongjia Feng
  • In-Fah M. Lee
  • Joerg D. Schulzke
  • Daniel H. Teitelbaum
Original Article



Total parenteral nutrition (TPN), a necessary treatment for patients who cannot receive enteral nutrition, is associated with infectious complications due in part to a loss of intestinal epithelial barrier function (EBF). Using a mouse model of TPN, with enteral nutrient deprivation, we previously demonstrated an increase in mucosal interferon-γ and tumor necrosis factor-α; these cytokine changes are a major mediator driving a reduction in epithelial tight junction (TJ) protein expression. However, the exact ultrastructural changes to the intestinal epithelial barrier have not been previously described.


We hypothesized that TPN dependence results in ultrastructural changes in the intestinal epithelial TJ meshwork.


C57BL/6 mice underwent internal jugular venous cannulation and were given enteral nutrition or TPN with enteral nutrient deprivation for 7 days. Freeze-fracture electron microscopy was performed on ileal tissue to characterize changes in TJ ultrastructure. EBF was measured using transepithelial resistance and tracer permeability, while TJ expression was measured via Western immunoblotting and immunofluorescence staining.


While strand density, linearity, and appearance were unchanged, TPN dependence led to a mean reduction in one horizontal strand out of the TJ compact meshwork to a more basal region, resulting in a reduction in meshwork depth. These findings were correlated with the loss of TJ localization of claudin-4 and tricellulin, reduced expression of claudin-5 and claudin-8, and reduced ex vivo EBF.


Tight junction ultrastructural changes may contribute to reduced EBF in the setting of TPN dependence.


Small intestine Parenteral nutrition Epithelial barrier function Tight junction Freeze-fracture electron microscopy 



This work was supported by the US National Institutes of Health (NIH), National Institute of Allergy and Infectious Diseases 5R01AI-44076-15 (to DHT).

Compliance with ethical standards

Conflict of interest



  1. 1.
    Abunnaja S, Cuviello A, Sanchez JA. Enteral and parenteral nutrition in the perioperative period: state of the art. Nutrients. 2013;5:608–623.CrossRefPubMedPubMedCentralGoogle Scholar
  2. 2.
    Moore FA, Feliciano DV, Andrassy RJ, et al. Early enteral feeding, compared with parenteral, reduces postoperative septic complications. The results of a meta-analysis. Ann Surg. 1992;216:172–183.CrossRefPubMedPubMedCentralGoogle Scholar
  3. 3.
    Braunschweig CL, Levy P, Sheean PM, Wang X. Enteral compared with parenteral nutrition: a meta-analysis. Am J Clin Nutr. 2001;74:534–542.PubMedGoogle Scholar
  4. 4.
    Peter JV, Moran JL, Phillips-Hughes J. A metaanalysis of treatment outcomes of early enteral versus early parenteral nutrition in hospitalized patients. Crit Care Med. 2005;33:213–220. (discussion 60-1).CrossRefPubMedGoogle Scholar
  5. 5.
    Squires RH, Duggan C, Teitelbaum DH, et al. Natural history of pediatric intestinal failure: initial report from the pediatric intestinal failure consortium. J Pediatr. 2012;161:723–728.CrossRefPubMedPubMedCentralGoogle Scholar
  6. 6.
    Buchman AL, Moukarzel AA, Bhuta S, et al. Parenteral nutrition is associated with intestinal morphologic and functional changes in humans. JPEN J Parenter Enteral Nutr. 1995;19:453–460.CrossRefPubMedGoogle Scholar
  7. 7.
    Alverdy JC, Aoys E, Moss GS. Total parenteral nutrition promotes bacterial translocation from the gut. Surgery. 1988;104:185–190.PubMedGoogle Scholar
  8. 8.
    Demehri FR, Barrett M, Ralls MW, Miyasaka EA, Feng Y, Teitelbaum DH. Intestinal epithelial cell apoptosis and loss of barrier function in the setting of altered microbiota with enteral nutrient deprivation. Front Cell Infect Microbiol. 2013;3:105.CrossRefPubMedPubMedCentralGoogle Scholar
  9. 9.
    Ralls MW, Demehri FR, Feng Y, et al. Enteral nutrient deprivation in patients leads to a loss of intestinal epithelial barrier function. Surgery. 2015;157:732–742.CrossRefPubMedPubMedCentralGoogle Scholar
  10. 10.
    Amasheh M, Grotjohann I, Amasheh S, et al. Regulation of mucosal structure and barrier function in rat colon exposed to tumor necrosis factor alpha and interferon gamma in vitro: a novel model for studying the pathomechanisms of inflammatory bowel disease cytokines. Scand J Gastroenterol. 2009;44:1226–1235.CrossRefPubMedGoogle Scholar
  11. 11.
    Menard S, Lebreton C, Schumann M, et al. Paracellular versus transcellular intestinal permeability to gliadin peptides in active celiac disease. Am J Pathol. 2012;180:608–615.CrossRefPubMedGoogle Scholar
  12. 12.
    Schumann M, Kamel S, Pahlitzsch ML, et al. Defective tight junctions in refractory celiac disease. Ann NY Acad Sci. 2012;1258:43–51.CrossRefPubMedGoogle Scholar
  13. 13.
    Sun X, Yang H, Nose K, et al. Decline in intestinal mucosal IL-10 expression and decreased intestinal barrier function in a mouse model of total parenteral nutrition. Am J Physiol Gastroint Liver Physiol. 2008;294:G139–G147.CrossRefGoogle Scholar
  14. 14.
    Feng Y, Sun X, Yang H, Teitelbaum DH. Dissociation of E-cadherin and beta-catenin in a mouse model of total parenteral nutrition: a mechanism for the loss of epithelial cell proliferation and villus atrophy. J Physiol. 2009;587:641–654.CrossRefPubMedPubMedCentralGoogle Scholar
  15. 15.
    Yang H, Kiristioglu I, Fan Y, et al. Interferon-gamma expression by intraepithelial lymphocytes results in a loss of epithelial barrier function in a mouse model of total parenteral nutrition. Ann Surg. 2002;236:226–234.CrossRefPubMedPubMedCentralGoogle Scholar
  16. 16.
    Yang H, Fan Y, Teitelbaum DH. Intraepithelial lymphocyte-derived interferon-gamma evokes enterocyte apoptosis with parenteral nutrition in mice. Am J Physiol Gastrointest Liver Physiol. 2003;284:G629–G637.CrossRefPubMedGoogle Scholar
  17. 17.
    Feng Y, Teitelbaum DH. Epidermal growth factor/TNF-alpha transactivation modulates epithelial cell proliferation and apoptosis in a mouse model of parenteral nutrition. Am J Physiol Gastrointest Liver Physiol. 2012;302:G236–G249.CrossRefPubMedPubMedCentralGoogle Scholar
  18. 18.
    Feng Y, Teitelbaum DH. Tumour necrosis factor-induced loss of intestinal barrier function requires TNFR1 and TNFR2 signalling in a mouse model of total parenteral nutrition. J Physiol. 2013;591:3709–3723.CrossRefPubMedPubMedCentralGoogle Scholar
  19. 19.
    Shen L, Weber CR, Raleigh DR, Yu D, Turner JR. Tight junction pore and leak pathways: a dynamic duo. Annu Rev Physiol. 2011;73:283–309.CrossRefPubMedPubMedCentralGoogle Scholar
  20. 20.
    Blikslager AT, Moeser AJ, Gookin JL, Jones SL, Odle J. Restoration of barrier function in injured intestinal mucosa. Physiol Rev. 2007;87:545–564.CrossRefPubMedGoogle Scholar
  21. 21.
    Gunzel D, Yu AS. Claudins and the modulation of tight junction permeability. Physiol Rev. 2013;93:525–569.CrossRefPubMedPubMedCentralGoogle Scholar
  22. 22.
    Aijaz S, Balda MS, Matter K. Tight junctions: molecular architecture and function. Int Rev Cytol. 2006;248:261–298.CrossRefPubMedGoogle Scholar
  23. 23.
    Mitic LL, Anderson JM. Molecular architecture of tight junctions. Annu Rev Physiol. 1998;60:121–142.CrossRefPubMedGoogle Scholar
  24. 24.
    Mitic LL, Van Itallie CM, Anderson JM. Molecular physiology and pathophysiology of tight junctions I. Tight junction structure and function: lessons from mutant animals and proteins. Am J Physiol Gastrointest Liver Physiol. 2000;279:G250–G254.PubMedGoogle Scholar
  25. 25.
    Krug SM, Schulzke JD, Fromm M. Tight junction, selective permeability, and related diseases. Semin Cell Dev Biol. 2014;36:166–176.CrossRefPubMedGoogle Scholar
  26. 26.
    Shen L, Black ED, Witkowski ED, et al. Myosin light chain phosphorylation regulates barrier function by remodeling tight junction structure. J Cell Sci. 2006;119:2095–2106.CrossRefPubMedGoogle Scholar
  27. 27.
    Petecchia L, Sabatini F, Usai C, Caci E, Varesio L, Rossi GA. Cytokines induce tight junction disassembly in airway cells via an EGFR-dependent MAPK/ERK1/2-pathway. Lab Invest. 2012;92:1140–1148.CrossRefPubMedGoogle Scholar
  28. 28.
    Hering NA, Fromm M, Schulzke JD. Determinants of colonic barrier function in inflammatory bowel disease and potential therapeutics. J Physiol. 2012;590:1035–1044.CrossRefPubMedPubMedCentralGoogle Scholar
  29. 29.
    Mankertz J, Schulzke JD. Altered permeability in inflammatory bowel disease: pathophysiology and clinical implications. Curr Opin Gastroenterol. 2007;23:379–383.CrossRefPubMedGoogle Scholar
  30. 30.
    Noth R, Lange-Grumfeld J, Stuber E, et al. Increased intestinal permeability and tight junction disruption by altered expression and localization of occludin in a murine graft versus host disease model. BMC Gastroenterol. 2011;11:109.CrossRefPubMedPubMedCentralGoogle Scholar
  31. 31.
    Watson AJ, Hughes KR. TNF-alpha-induced intestinal epithelial cell shedding: implications for intestinal barrier function. Ann NY Acad Sci. 2012;1258:1–8.CrossRefPubMedGoogle Scholar
  32. 32.
    Wang F, Graham WV, Wang Y, Witkowski ED, Schwarz BT, Turner JR. Interferon-gamma and tumor necrosis factor-alpha synergize to induce intestinal epithelial barrier dysfunction by up-regulating myosin light chain kinase expression. Am J Pathol. 2005;166:409–419.CrossRefPubMedPubMedCentralGoogle Scholar
  33. 33.
    Williams JM, Duckworth CA, Watson AJ, et al. A mouse model of pathological small intestinal epithelial cell apoptosis and shedding induced by systemic administration of lipopolysaccharide. Dis Model Mech. 2013;6:1388–1399.CrossRefPubMedPubMedCentralGoogle Scholar
  34. 34.
    Ye D, Ma I, Ma TY. Molecular mechanism of tumor necrosis factor-alpha modulation of intestinal epithelial tight junction barrier. Am J Physiol Gastrointest Liver Physiol. 2006;290:G496–G504.CrossRefPubMedGoogle Scholar
  35. 35.
    Marchiando AM, Shen L, Graham WV, et al. The epithelial barrier is maintained by in vivo tight junction expansion during pathologic intestinal epithelial shedding. Gastroenterology. 2011;140:1208–1218.e1-2.CrossRefPubMedPubMedCentralGoogle Scholar
  36. 36.
    Cunningham KE, Turner JR. Myosin light chain kinase: pulling the strings of epithelial tight junction function. Ann NY Acad Sci. 2012;1258:34–42.CrossRefPubMedPubMedCentralGoogle Scholar
  37. 37.
    Chen C, Wang P, Su Q, Wang S, Wang F. Myosin light chain kinase mediates intestinal barrier disruption following burn injury. PLoS ONE. 2012;7:e34946.CrossRefPubMedPubMedCentralGoogle Scholar
  38. 38.
    Severs NJ. Freeze-fracture electron microscopy. Nat Protoc. 2007;2:547–576.CrossRefPubMedGoogle Scholar
  39. 39.
    Zeissig S, Burgel N, Gunzel D, et al. Changes in expression and distribution of claudin 2, 5 and 8 lead to discontinuous tight junctions and barrier dysfunction in active Crohn’s disease. Gut. 2007;56:61–72.CrossRefPubMedPubMedCentralGoogle Scholar
  40. 40.
    Muza-Moons MM, Schneeberger EE, Hecht GA. Enteropathogenic Escherichia coli infection leads to appearance of aberrant tight junctions strands in the lateral membrane of intestinal epithelial cells. Cell Microbiol. 2004;6:783–793.CrossRefPubMedGoogle Scholar
  41. 41.
    Kiristioglu I, Antony P, Fan Y, et al. Total parenteral nutrition-associated changes in mouse intestinal intraepithelial lymphocytes. Dig Dis Sci. 2002;47:1147–1157.CrossRefPubMedGoogle Scholar
  42. 42.
    Yang H, Feng Y, Sun X, Teitelbaum DH. Enteral versus parenteral nutrition: effect on intestinal barrier function. Ann NY Acad Sci. 2009;1165:338–346.CrossRefPubMedPubMedCentralGoogle Scholar
  43. 43.
    Feng Y, McDunn JE, Teitelbaum DH. Decreased phospho-Akt signaling in a mouse model of total parenteral nutrition: a potential mechanism for the development of intestinal mucosal atrophy. Am J Physiol Gastrointest Liver Physiol. 2010;298:G833–G841.CrossRefPubMedPubMedCentralGoogle Scholar
  44. 44.
    Kiristioglu I, Teitelbaum DH. Alteration of the intestinal intraepithelial lymphocytes during total parenteral nutrition. J Surg Res. 1998;79:91–96.CrossRefPubMedGoogle Scholar
  45. 45.
    Nose K, Yang H, Sun X, et al. Glutamine prevents total parenteral nutrition-associated changes to intraepithelial lymphocyte phenotype and function: a potential mechanism for the preservation of epithelial barrier function. J Interferon Cytokine Res. 2010;30:67–80.CrossRefPubMedPubMedCentralGoogle Scholar
  46. 46.
    Grossmann J, Maxson JM, Whitacre CM, et al. New isolation technique to study apoptosis in human intestinal epithelial cells. Am J Pathol. 1998;153:53–62.CrossRefPubMedPubMedCentralGoogle Scholar
  47. 47.
    Schwarz BT, Wang F, Shen L, et al. LIGHT signals directly to intestinal epithelia to cause barrier dysfunction via cytoskeletal and endocytic mechanisms. Gastroenterology. 2007;132:2383–2394.CrossRefPubMedPubMedCentralGoogle Scholar
  48. 48.
    Stevenson BR, Anderson JM, Goodenough DA, Mooseker MS. Tight junction structure and ZO-1 content are identical in two strains of Madin-Darby canine kidney cells which differ in transepithelial resistance. J Cell Biol. 1988;107:2401–2408.CrossRefPubMedGoogle Scholar
  49. 49.
    Suzuki F, Nagano T. Three-dimensional model of tight junction fibrils based on freeze-fracture images. Cell Tissue Res. 1991;264:381–384.CrossRefPubMedGoogle Scholar
  50. 50.
    Reims A, Strandvik B, Sjövall H. Epithelial electrical resistance as a measure of permeability changes in pediatric duodenal biopsies. J Pediatr Gastroenterol Nutr. 2006;43:619–623.CrossRefPubMedGoogle Scholar
  51. 51.
    Ikenouchi J, Furuse M, Furuse K, Sasaki H, Tsukita S, Tsukita S. Tricellulin constitutes a novel barrier at tricellular contacts of epithelial cells. J Cell Biol. 2005;171:939–945.CrossRefPubMedPubMedCentralGoogle Scholar
  52. 52.
    Casaer MP, Mesotten D, Hermans G, et al. Early versus late parenteral nutrition in critically ill adults. N Engl J Med. 2011;365:506–517.CrossRefPubMedGoogle Scholar
  53. 53.
    Pfuntner A, Wier LM, Stocks C. Most Frequent Procedures Performed in U.S. Hospitals, 2010: Statistical Brief #149. Rockville: Healthcare Cost and Utilization Project (HCUP) Statistical Briefs; 2006.Google Scholar
  54. 54.
    Spencer AU, Kovacevich D, McKinney-Barnett M, et al. Pediatric short-bowel syndrome: the cost of comprehensive care. Am J Clin Nutr. 2008;88:1552–1559.CrossRefPubMedGoogle Scholar
  55. 55.
    Kansagra K, Stoll B, Rognerud C, et al. Total parenteral nutrition adversely affects gut barrier function in neonatal piglets. Am J Physiol Gastrointest Liver Physiol. 2003;285:G1162–G1170.CrossRefPubMedGoogle Scholar
  56. 56.
    Buzby GP. Overview of randomized clinical trials of total parenteral nutrition for malnourished surgical patients. World J Surg. 1993;17:173–177.CrossRefPubMedGoogle Scholar
  57. 57.
    Wildhaber BE, Yang H, Spencer AU, Drongowski RA, Teitelbaum DH. Lack of enteral nutrition—effects on the intestinal immune system. J Surg Res. 2005;123:8–16.CrossRefPubMedGoogle Scholar
  58. 58.
    Schulzke JD, Ploeger S, Amasheh M, et al. Epithelial tight junctions in intestinal inflammation. Ann NY Acad Sci. 2009;1165:294–300.CrossRefPubMedGoogle Scholar
  59. 59.
    Meddings J. Barrier dysfunction and Crohn’s disease. Ann NY Acad Sci. 2000;915:333–338.CrossRefPubMedGoogle Scholar
  60. 60.
    Marin ML, Greenstein AJ, Geller SA, Gordon RE, Aufses AH Jr. A freeze fracture study of Crohn’s disease of the terminal ileum: changes in epithelial tight junction organization. Am J Gastroenterol. 1983;78:537–547.PubMedGoogle Scholar
  61. 61.
    Schulzke JD, Bojarski C, Zeissig S, Heller F, Gitter AH, Fromm M. Disrupted barrier function through epithelial cell apoptosis. Ann NY Acad Sci. 2006;1072:288–299.CrossRefPubMedGoogle Scholar
  62. 62.
    Zeissig S, Bojarski C, Buergel N, et al. Downregulation of epithelial apoptosis and barrier repair in active Crohn’s disease by tumour necrosis factor alpha antibody treatment. Gut. 2004;53:1295–1302.CrossRefPubMedPubMedCentralGoogle Scholar
  63. 63.
    Van Itallie C, Rahner C, Anderson JM. Regulated expression of claudin-4 decreases paracellular conductance through a selective decrease in sodium permeability. J Clin Invest. 2001;107:1319–1327.CrossRefPubMedPubMedCentralGoogle Scholar
  64. 64.
    Colegio OR, Van Itallie CM, McCrea HJ, Rahner C, Anderson JM. Claudins create charge-selective channels in the paracellular pathway between epithelial cells. Am J Physiol Cell Physiol. 2002;283:C142–C147.CrossRefPubMedGoogle Scholar
  65. 65.
    Yang H, Finaly R, Teitelbaum DH. Alteration in epithelial permeability and ion transport in a mouse model of total parenteral nutrition. Crit Care Med. 2003;31:1118–1125.CrossRefPubMedGoogle Scholar
  66. 66.
    Xiao W, Feng Y, Holst JJ, Hartmann B, Yang H, Teitelbaum DH. Glutamate prevents intestinal atrophy via luminal nutrient sensing in a mouse model of total parenteral nutrition. FASEB J. 2014;28:2073–2087.CrossRefPubMedPubMedCentralGoogle Scholar
  67. 67.
    Oda Y, Otani T, Ikenouchi J, Furuse M. Tricellulin regulates junctional tension of epithelial cells at tricellular contacts through Cdc42. J Cell Sci. 2014;127:4201–4212.CrossRefPubMedGoogle Scholar
  68. 68.
    Krug SM, Amasheh S, Richter JF, et al. Tricellulin forms a barrier to macromolecules in tricellular tight junctions without affecting ion permeability. Mol Biol Cell. 2009;20:3713–3724.CrossRefPubMedPubMedCentralGoogle Scholar
  69. 69.
    Krug SM, Amasheh M, Dittmann I, Christoffel I, Fromm M, Amasheh S. Sodium caprate as an enhancer of macromolecule permeation across tricellular tight junctions of intestinal cells. Biomaterials. 2013;34:275–282.CrossRefPubMedGoogle Scholar
  70. 70.
    Zakrzewski SS, Richter JF, Krug SM, et al. Improved cell line IPEC-J2, characterized as a model for porcine jejunal epithelium. PLoS ONE. 2013;8:e79643.CrossRefPubMedPubMedCentralGoogle Scholar
  71. 71.
    Claude P, Goodenough DA. Fracture faces of zonulae occludentes from “tight” and “leaky” epithelia. J Cell Biol. 1973;58:390–400.CrossRefPubMedPubMedCentralGoogle Scholar
  72. 72.
    Claude P. Morphological factors influencing transepithelial permeability: a model for the resistance of the zonula occludens. J Membr Biol. 1978;39:219–232.CrossRefPubMedGoogle Scholar

Copyright information

© Springer Science+Business Media New York 2015

Authors and Affiliations

  1. 1.Section of Pediatric Surgery, Department of SurgeryMott Children’s Hospital, University of Michigan Health SystemAnn ArborUSA
  2. 2.Institute of Clinical PhysiologyCharité – Universitätsmedizin BerlinBerlinGermany

Personalised recommendations