Ex Vivo Intestinal Sacs at vurdere slimhinder permeabilitet i modeller af gastrointestinal sygdom
Summary
This protocol describes the use of excised intestinal tissue preparations or “intestinal sacs” as an ex vivo model of intestinal barrier function. This model may be used to assess integrity of both the epithelial barrier and the mucous gel layer at specific intestinal sites in animal models of digestive disease.
Abstract
Epitelbarrieren er den første medfødte forsvar af mave-tarmkanalen og selektivt regulerer transport fra lumenet til det underliggende væv rum, der begrænser transport af mindre molekyler over slimhinder og næsten fuldstændigt forbud epitelial makromolekylær transport. Denne selektivitet er bestemt af den mukøse gellaget, som begrænser transporten af lipofile molekyler og begge de apikale receptorer og stramme forbindelsesepitoper proteinkomplekser af epitelet. In vitro-cellekultur modeller af epitelet er bekvemme, men som en model, mangler de kompleksitet samspillet mellem mikrobiota, slim-gel, epitel og immunsystemet. På den anden side, kan udføres in vivo vurdering af intestinal absorption eller permeabilitet, men disse assays måler den overordnede gastrointestinal absorption, uden angivelse af site-specificitet. Ex vivo permeabilitet assays ved hjælp af "intestinale sække"; er en hurtig og følsom metode til måling enten samlet intestinal integritet eller sammenlignende transport af et specifikt molekyle, med den ekstra fordel af intestinal websted specificitet. Her beskriver vi udarbejdelsen af intestinale sække for permeabilitet undersøgelser og beregning af den tilsyneladende permeabilitet (P app) af et molekyle gennem tarmbarrieren. Denne teknik kan anvendes som en metode til vurdering lægemiddelabsorption, eller undersøge regional epitelbarriere dysfunktion i dyremodeller af gastrointestinal sygdom.
Introduction
Tarmens epitelbarriere af mave-tarmkanalen er en slimhindeoverflade område anslås til 400 m 2 i den humane voksne. Følgelig er det konstant udsat for slås fra mikrober, indtaget lægemidler, næringsstoffer og bakterielle toksiner. Værten skal ikke kun skelne mellem tolerable kommensale bakterier og potentielle patogener, men skal forhindre disse arter og deres udskilte molekyler fra kryds epitelbarrieren, mens den på samme tid tillader absorption af næringsstoffer. Således rolle tarmepitelet er at fungere som en selektiv barriere til de luminale indhold 1. Dette opnås til dels ved den medfødte epitel- forsvarssystem ved slimhinden, som virker gennem en responsiv biologisk system bestående af konstitutive og inducerbare mekanismer 2.
Tab af Epitelbarrierefunktionen er en patologi, der er karakteristisk for en række gastrointestinale sygdomme. In vivoundersøgelse af epitelial barrierefunktion kan vurderes ved oral sonde af et sporstof molekyle og efterfølgende serum analyse 3. Denne teknik giver ingen indikation med hensyn til stedet for barriere dysfunktion. In vitro og ex vivo evaluering af transepitel modstand ved hjælp Transwell systemer 3 og Ussing kamrene 4,5 henholdsvis er almindeligt ansat som surrogatmarkører for epitelial barrierefunktion, men mangler de bidragydende sygdom fysiologi dyremodeller 6. I denne protokol beskriver vi en ex vivo vævspræparat model, der tillader direkte og lokaliseret vurdering af intestinal integritet, og som kan anvendes til at vurdere mucosal barrierefunktion ved en række niveauer. Vigtigt er det, kan denne teknik anvendes på dyremodeller for sygdom, eller kan være farmakologisk manipuleres for at muliggøre en grundig søgning i slimhindebarrieren dysfunktion.
Protocol
Representative Results
Discussion
Her har vi beskrevet isolering og fremstilling af intestinale sække til vurdering mucosal barrierefunktion ex vivo. Intestinal sac præparater er primært anvendt i farmaceutisk forskning, undersøge absorptionen af lægemiddelkandidater i hele tarmen. Imidlertid er dette assay lige velegnet til undersøgelse af intestinal sygdom. Intestinal permeabilitet kan variere meget fra område til område og stedet konkret vurdering af permeabilitet giver en bedre forståelse af den regionale betydning af slimhin…
Divulgations
The authors have nothing to disclose.
Acknowledgements
This work was funded by National Health and Medical Research Project Grant APP1021582 and a Hunter Medical Research Institute grant sponsored by Sparke Helmore/NBN Triathlon and the Estate of the late Leslie Kenneth McFarlane.
Materials
| Dekantel Non-absorbable Silk suture | Braintree Scientific | SUT-S 116 | |
| Media 199 (TC199) | Life Technologies | 11043-023 | No phenol red as this interferes with fluorescence |
| Dulbecco's Modified Eagle Medium (DMEM) | Life Technologies | 21063-045 | No phenol red as this interferes with fluorescence |
| N-acetylcysteine | Sigma Aldrich | Use at 10mM in media | |
| Small animal vascular cathether: Physiocath | Data Sciences International | 277-1-002 | |
| FITC-Dextran 4400 MW | Sigma Aldrich | FD-4 | |
| FITC-Dextran 20,000 MW | Sigma Aldrich | FD-20 | |
| FITC-Dextran 70,000 MW | Sigma Aldrich | FD-70 |
References
- Goggins, B. J., Chaney, C., Radford-Smith, G. L., Horvat, J. C., Keely, S. Hypoxia and Integrin-Mediated Epithelial Restitution during Mucosal Inflammation. Frontiers in immunology. 4, 272 (2013).
- Otte, J. M., Kiehne, K., Herzig, K. H. Antimicrobial peptides in innate immunity of the human intestine. Journal of gastroenterology. 38, 717-726 (2003).
- Robinson, A., et al. Mucosal protection by hypoxia-inducible factor prolyl hydroxylase inhibition. Gastroenterology. 134, 145-155 (2008).
- Feighery, L., et al. Increased intestinal permeability in rats subjected to traumatic frontal lobe percussion brain injury. The Journal of trauma. 64, 131-137 (2008).
- Keely, S., et al. Chloride-led disruption of the intestinal mucous layer impedes Salmonella invasion: evidence for an ‘enteric tear’ mechanism. Cellular physiology and biochemistry : international journal of experimental cellular physiology, biochemistry, and pharmacology. 28, 743-752 (2011).
- Keely, S., et al. Contribution of epithelial innate immunity to systemic protection afforded by prolyl hydroxylase inhibition in murine colitis. Mucosal immunology. 7, 114-123 (2014).
- Sourisseau, T., et al. Regulation of PCNA and cyclin D1 expression and epithelial morphogenesis by the ZO-1-regulated transcription factor ZONAB/DbpA. Mol Cell Biol. 26, 2387-2398 (2006).
- Ruehl-Fehlert, C., et al. Revised guides for organ sampling and trimming in rats and mice–part 1. Exp Toxicol Pathol. 55, 91-106 (2003).
- Barthe, L., Woodley, J. F., Kenworthy, S., Houin, G. An improved everted gut sac as a simple and accurate technique to measure paracellular transport across the small intestine. European journal of drug metabolism and pharmacokinetics. 23, 313-323 (1998).
- Marks, E., et al. Oral Delivery of Prolyl Hydroxylase Inhibitor: AKB-4924 Promotes Localized Mucosal Healing in a Mouse Model of Colitis. Inflammatory bowel diseases. 21, 267-275 (2015).
- Keely, S., et al. Hypoxia-inducible factor-dependent regulation of platelet-activating factor receptor as a route for gram-positive bacterial translocation across epithelia. Mol Biol Cell. 21, 538-546 (2010).
- Brayden, D. J., Bzik, V. A., Lewis, A. L., Illum, L. CriticalSorb promotes permeation of flux markers across isolated rat intestinal mucosae and Caco-2 monolayers. Pharmaceutical research. 29, 2543-2554 (2012).
- Hubbard, D., Ghandehari, H., Brayden, D. J. Transepithelial transport of PAMAM dendrimers across isolated rat jejunal mucosae in ussing chambers. Biomacromolecules. 15, 2889-2895 (2014).
- Keely, S., et al. In vitro and ex vivo intestinal tissue models to measure mucoadhesion of poly (methacrylate) and N-trimethylated chitosan polymers. Pharmaceutical research. 22, 38-49 (2005).
- Maher, S., et al. Evaluation of intestinal absorption enhancement and local mucosal toxicity of two promoters. I. Studies in isolated rat and human colonic mucosae. European journal of pharmaceutical sciences : official journal of the European Federation for Pharmaceutical Sciences. 38, 291-300 (2009).
- Balda, M. S., et al. Functional dissociation of paracellular permeability and transepithelial electrical resistance and disruption of the apical-basolateral intramembrane diffusion barrier by expression of a mutant tight junction membrane protein. The Journal of cell biology. 134, 1031-1049 (1996).
- Behrens, I., Stenberg, P., Artursson, P., Kissel, T. Transport of lipophilic drug molecules in a new mucus-secreting cell culture model based on HT29-MTX cells. Pharmaceutical research. 18, 1138-1145 (2001).
- Stefka, A. T., et al. Commensal bacteria protect against food allergen sensitization. Proc Natl Acad Sci U S A. 111, 13145-13150 (2014).
- Keely, S., et al. Activated fluid transport regulates bacterial-epithelial interactions and significantly shifts the murine colonic microbiome. Gut microbes. 3, 250-260 (2012).
- Barrett, K. E., Keely, S. J. Chloride secretion by the intestinal epithelium: molecular basis and regulatory aspects. Annual review of physiology. 62, 535-572 (2000).
- Soni, J., et al. Rat, ovine and bovine Peyer’s patches mounted in horizontal diffusion chambers display sampling function. Journal of controlled release : official journal of the Controlled Release Society. 115, 68-77 (2006).
- Justino, P. F., et al. Regulatory role of Lactobacillus acidophilus on inflammation and gastric dysmotility in intestinal mucositis induced by 5-fluorouracil in mice. Cancer chemotherapy and pharmacology. , (2015).
- Tran, C. D., Sundar, S., Howarth, G. S. Dietary zinc supplementation and methotrexate-induced small intestinal mucositis in metallothionein-knockout and wild-type mice. Cancer biology & therapy. 8, 1662-1667 (2009).
- Musch, M. W., Wang, Y., Claud, E. C., Chang, E. B. Lubiprostone decreases mouse colonic inner mucus layer thickness and alters intestinal microbiota. Digestive diseases and sciences. 58, 668-677 (2013).
