JoVE Logo
Faculty Resource Center

Sign In

Summary

Abstract

Introduction

Protocol

Representative Results

Discussion

Acknowledgements

Materials

References

Medicine

An In Vivo Method for Evaluating the Gut-Blood Barrier and Liver Metabolism of Microbiota Products

Published: October 20th, 2018

DOI:

10.3791/58456

1Department of Experimental Physiology and Pathophysiology, Laboratory of Centre for Preclinical Research, Medical University of Warsaw, 2Mass Spectrometry Laboratory, Institute of Biochemistry and Biophysics, Polish Academy of Sciences

The access of nutrients, microbiota metabolites and medicines to the circulation is controlled by the gut-blood barrier (GBB). We describe a direct method for measuring the GBB permeability in vivo, which, in contrast to commonly used indirect methods, is virtually not affected by liver and kidney functions.

The gut-blood barrier (GBB) controls the passage of nutrients, bacterial metabolites and drugs from intestinal lumen to the bloodstream. The GBB integrity is disturbed in gastrointestinal, cardiovascular and metabolic diseases, which may result in easier access of biologically active compounds, such as gut bacterial metabolites, to the bloodstream. Thus, the permeability of the GBB may be a marker of both intestinal and extraintestinal diseases. Furthermore, the increased penetration of bacterial metabolites may affect the functioning of the entire organism.

Commonly used methods for studying the GBB permeability are performed ex vivo. The accuracy of those methods is limited, because the functioning of the GBB depends on intestinal blood flow. On the other hand, commonly used in vivo methods may be biased by liver and kidney performance, as those methods are based on evaluation of urine or/and peripheral blood concentrations of exogenous markers. Here, we present a direct measurement of GBB permeability in rats using an in vivo method based on portal blood sampling, which preserves intestinal blood flow and is virtually not affected by the liver and kidney function.

Polyurethane catheters are inserted into the portal vein and inferior vena cava just above the hepatic veins confluence. Blood is sampled at baseline and after administration of a selected marker into a desired part of the gastrointestinal tract. Here, we present several applications of the method including (1) evaluation of the colon permeability to TMA, a gut bacterial metabolite, (2) evaluation of liver clearance of TMA, and (3) evaluation of a gut-portal blood-liver-peripheral blood pathway of gut bacteria-derived short-chain fatty acids. Furthermore, the protocol may also be used for tracking intestinal absorption and liver metabolism of drugs or for measurements of portal blood pressure.

The gut-blood barrier (GBB), also known as the intestinal barrier, is a complex multilayer system that separates the gut lumen from the bloodstream in order to limit the passage of harmful compounds while allowing the absorption of nutrients1. It consists of the three main layers: the mucus layer, epithelium and lamina propria.

Numerous factors may affect the GBB integrity and function2. It has been shown that GBB is disturbed in both gastrointestinal and extraintestinal diseases, including cardiovascular and metabolic diseases3, which may lead to an increased passage o....

Log in or to access full content. Learn more about your institution’s access to JoVE content here

The experiments were performed on male Wistar Kyoto rats according to Directive 2010/63 EU on the protection of animals used for scientific purposes and were approved by the I Local Bioethical Committee in Warsaw.

1. Insertion of the Line for Intraintestinal Administration

NOTE: Here we propose intracolonic administration of a marker using a catheter. It may be modified by oral administration or gavage at various levels of the digestive tract e.g........

Log in or to access full content. Learn more about your institution’s access to JoVE content here

We have successfully measured the GBB permeability and liver clearance of TMA in rats. We have demonstrated that hypertensive rats have an increased colon permeability to TMA in comparison to normotensive rats (Figure 2)4. In another study we found that high salt intake does not affect the GBB permeability and liver clearance of TMA (Figure 3)14.

Log in or to access full content. Learn more about your institution’s access to JoVE content here

The described direct, in vivo, method of measuring the GBB permeability maintains closetophysiological conditions in the gastrointestinal system (preserves the intestinal blood flow), and is virtually not influenced by liver and kidney function.

The critical step of this technique is the insertion of the portal catheter. This must be done gently and decisively at the same time. A mild, short bleeding may occur from the correctly performed puncture of the portal vein; however, it stops.......

Log in or to access full content. Learn more about your institution’s access to JoVE content here

The work is supported by the Ministry of Science and Higher Education Republic of Poland, Diamond grant no: DI2017 009247.

....

Log in or to access full content. Learn more about your institution’s access to JoVE content here

Name Company Catalog Number Comments
Needle OD: 9 mm Becton Dickinson  S.A. 301300
Polyethylene catheter ID: 0.025", OD: 0.040"  Scientific Commodities, Inc. #BB520-40
Polyethylene catheter ID: 0.012", OD: 0.025"  Scientific Commodities, Inc. #BB520-25
C-Flex Tubing,Opaque White 1/50"ID x 1/12 " OD   Cole-Parmer Instrument Co. 06424-59
Pediatric Foley catheter (size 10F or 8F)  Sigmed 0000 80305
Surgical ligatures 3/0 Yavo Sp. Z o.o.  P48JE
Absorbable surgical sutures - Polyglactine 910 4/0 KRUUSE Polska Sp. Zo.o. 152336
Tissue glue - Loctite 454Cyanoacrylate Adhesive Loctite  1370127
Povidone iodine EGIS Pharmaceuticals PLC 4449 11
Heparin - Heparinium WZF  WZF Polfa S.A. 02BK0417 Dilute 10 times with physiological saline
Glycerin 86% Laboratorium Farmaceutyczne Avena 5.90999E+12 Serves as a lubricant in colon catheterization
Xylocaine 2% AstraZenca  9941342
Urethane Sigma-Aldrich (Merck)  U2500-500G
Trimethylamine solution 45% Sigma-Aldrich (Merck)  92262-1L
Syringes 2 mL B.Braun Melsungen AG 4606027V
Saline 250 mL Fraesenius Kabi Polska Sp. Z o.o. 15LL707WL
Surgical scissors, straight, length 115 mm, 4 1/2 "blunt ends Braun NS-010-115-PKM
Artery forceps type Micro-Adson bent, length 140 mm 5 1/2 " Braun KN-008-140-ZMK
Anatomic forceps, lenght 95 mm, 3 3/4" sharp 0.7x0.55 Braun PO-001-007-ZMK
Micro Scissors type Vannas,  straight, lenght 85 mm, 3 3/8 " the length of the blades 6 mm Braun  NO-010-085-PMK
Towel clamps type Backhouse, lenght 130 mm, 5 1/8"  Braun HO-128-130-PMK
Needle holders, lenght 150 mm, 6" t=0.4 1/2  Braun  IM-927-150-PZMK
Delicate Scissors, lenght 110 mm , straight, 4 3/8” sharp  Braun NO-052-110-PMK
Anatomic forceps, lenght 95 mm, 3 3/4" sharp Braun PO-022-001-PMK

  1. Camilleri, M., Madsen, K., Spiller, R., Greenwood-Van Meerveld, B., Verne, G. N. Intestinal barrier function in health and gastrointestinal disease. Neurogastroenterology and Motility: The Official Journal of the European Gastrointestinal Motility Society. 24 (6), 503-512 (2012).
  2. Keita, A. V., Soderholm, J. D. The intestinal barrier and its regulation by neuroimmune factors. Neurogastroenterology and Motility: The Official Journal of the European Gastrointestinal Motility Society. 22 (7), 718-733 (2010).
  3. Farhadi, A., Banan, A., Fields, J., Keshavarzian, A. Intestinal barrier: an interface between health and disease. Journal of Gastroenterology and Hepatology. 18 (5), 479-497 (2003).
  4. Jaworska, K., et al. Hypertension in rats is associated with an increased permeability of the colon to TMA, a gut bacteria metabolite. PloS one. 12 (12), e0189310 (2017).
  5. Fujii, H., Nakai, K., Fukagawa, M. Role of oxidative stress and indoxyl sulfate in progression of cardiovascular disease in chronic kidney disease. Therapeutic Apheresis and Dialysis: Official Peer-Reviewed Journal of the International Society for Apheresis, the Japanese Society for Apheresis, the Japanese Society for Dialysis Therapy. 15 (2), 125-128 (2011).
  6. Tomasova, L., et al. Intracolonic hydrogen sulfide lowers blood pressure in rats. Nitric Oxide: Biology and Chemistry. 60, 50-58 (2016).
  7. Brahe, L. K., Astrup, A., Larsen, L. H. Is butyrate the link between diet, intestinal microbiota and obesity-related metabolic diseases?. Obesity Reviews: An Official Journal of the International Association for the Study of Obesity. 14 (12), 950-959 (2013).
  8. Ufnal, M., et al. Trimethylamine-N-oxide: a carnitine-derived metabolite that prolongs the hypertensive effect of angiotensin II in rats. The Canadian Journal of Cardiology. 30 (12), 1700-1705 (2014).
  9. Huc, T., Nowinski, A., Drapala, A., Konopelski, P., Ufnal, M. Indole and indoxyl sulfate, gut bacteria metabolites of tryptophan, change arterial blood pressure via peripheral and central mechanisms in rats. Pharmacological Research. 130, 172-179 (2018).
  10. Ufnal, M., Pham, K. The gut-blood barrier permeability - A new marker in cardiovascular and metabolic diseases?. Medical Hypotheses. 98, 35-37 (2017).
  11. Le Ferrec, E., et al. In vitro models of the intestinal barrier. The report and recommendations of ECVAM Workshop 46. European Centre for the Validation of Alternative methods. Alternatives to Laboratory Animals: ATLA. 29 (6), 649-668 (2001).
  12. Bohets, H., et al. Strategies for absorption screening in drug discovery and development. Current Topics in Medicinal Chemistry. 1 (5), 367-383 (2001).
  13. Grootjans, J., et al. Non-invasive assessment of barrier integrity and function of the human gut. World Journal of Gastrointestinal Surgery. 2 (3), 61-69 (2010).
  14. Bielinska, K., et al. High salt intake increases plasma trimethylamine N-oxide (TMAO) concentration and produces gut dysbiosis in rats. Nutrition. 54, 33-39 (2018).
  15. Clarke, L. L. A guide to Ussing chamber studies of mouse intestine. American journal of physiology. Gastrointestinal and Liver Physiology. 296 (6), G1151-G1166 (2009).
  16. Denno, D. M., et al. Use of the lactulose to mannitol ratio to evaluate childhood environmental enteric dysfunction: a systematic review. Clinical Infectious Diseases: An Official Publication of the Infectious Diseases Society of America. 59 Suppl 4, S213-S219 (2014).
  17. Lugea, A., Salas, A., Casalot, J., Guarner, F., Malagelada, J. R. Surface hydrophobicity of the rat colonic mucosa is a defensive barrier against macromolecules and toxins. Gut. 46 (4), 515-521 (2000).
  18. Bloemen, J. G., et al. Short chain fatty acids exchange across the gut and liver in humans measured at surgery. Clinical Nutrition. 28 (6), 657-661 (2009).
  19. Huc, T., et al. Colonic hydrogen sulfide produces portal hypertension and systemic hypotension in rats. Experimental Biology and Medicine. 243 (1), 96-106 (2018).

This article has been published

Video Coming Soon

JoVE Logo

Privacy

Terms of Use

Policies

Research

Education

ABOUT JoVE

Copyright © 2024 MyJoVE Corporation. All rights reserved