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In This Article

  • Summary
  • Abstract
  • Introduction
  • Protocol
  • Representative Results
  • Discussion
  • Acknowledgements
  • Materials
  • References
  • Reprints and Permissions

Summary

This protocol describes how to measure intestinal permeability of Caenorhabditis elegans. This method is helpful for basic biological research on intestinal health related to the interaction between intestinal bacteria and their host and for screening to identify probiotic and chemical agents to cure leaky gut syndrome and inflammatory bowel diseases.

Abstract

In living organisms, intestinal hyperpermeability is a serious symptom that leads to many inflammatory bowel diseases (IBDs). Caenorhabditis elegans is a nonmammalian animal model that is widely used as an assay system due to its short lifespan, transparency, cost-effectiveness, and lack of animal ethics issues. In this study, a method was developed to investigate the effects of different bacteria and 3,3'-diindolylmethane (DIM) on the intestinal permeability of C. elegans with a high-throughput image analysis system. The worms were infected with different gut bacteria or cotreated with DIM for 48 h and fed with fluorescein isothiocyanate (FITC)-dextran overnight. Then, the intestinal permeability was examined by comparing the fluorescence images and the fluorescence intensity inside the worm bodies. This method may also have the potential to identify probiotic and pathogenic intestinal bacteria that affect intestinal permeability in the animal model and is effective for examining the effects of harmful or health-promoting chemicals on intestinal permeability and intestinal health. However, this protocol also has some considerable limitations at the genetic level, especially for determining which genes are altered to control illness, because this method is mostly used for phenotypic determination. In addition, this method is limited to determining exactly which pathogenic substrates cause inflammation or increase the permeability of the worms' intestines during infection. Therefore, further in-depth studies, including investigation of the molecular genetic mechanism using mutant bacteria and nematodes as well as chemical component analysis of bacteria, are required to fully evaluate the function of bacteria and chemicals in determining intestinal permeability.

Introduction

Intestinal permeability is considered as one of the main barriers related to the intestinal microbiota and mucosal immunity and is likely to be affected by several factors, such as gut microbiota modifications, epithelial impairment, or mucus layer alterations1. Recent papers have reported effective protocols to measure the intestinal permeability of cultured human intestinal cells by analyzing the fluorescence flux rates across the intestinal cell layer2, but fewer research papers present a suitable procedure for measuring the gut permeability in nematodes, particularly in C. elegans, by using FITC-dextran stai....

Protocol

1. Preparation of P. aeruginosa PAO1 and Escherichia coli OP50 Culture

  1. Prepare 500 mL of sterilized Luria-Bertani (LB) medium (Table 1) and inoculate a single colony of P. aeruginosa into the medium. Incubate the culture for 14 to 15 h at 37 °C with a shaking speed of 150 rpm.
  2. Equally distribute the bacterial culture into two 500 mL centrifuge tubes and centrifuge the tubes at 3,220 x g at 4 °C for 30 min.
  3. Remove the supernatan.......

Representative Results

After incubation with P. aeruginosa PAO1, C. elegans showed a significant increase in FITC-dextran fluorescence in the worm body compared to the fluorescence shown after incubation with the other two bacterial strains (Figure 1). The fluorescence intensities of worms fed with E. coli OP50, P. aeruginosa PAO1, and E. faecalis KCTC3206 were 100.0 ± 6.6, 369.7 ± 38.9, and 105.6 ± 10.6%, respectively. The data emphasize that P. aeru.......

Discussion

By utilizing this new method for determining gut permeability in C. elegans, which combines automated fluorescence microscopy and quantitative image analysis, the differences caused by intestinal microorganisms or chemicals can be determined in vivo, specifically in the C. elegans intestine. This protocol is useful for gut permeability investigations and applicable to many tasks, such as reactive oxygen species (ROS) determination under stress conditions and morphological examinations, due to its conven.......

Acknowledgements

This study was supported by a Korea Institute of Science and Technology intramural research grant (2E29563).

....

Materials

NameCompanyCatalog NumberComments
3,3’-diindolylmethane SigmaD9568
90×15 mm Petri dishesSPL Life Sciences, South Korea10090
60×15 mm Petri dishesSPL Life Sciences, South Korea10060
Bactor AgarBeckton DickinsonREF. 214010
Formaldehyde solution SigmaF1635
Brain Heart Infusion (BHI) Becton DickinsonREF. 237500
Caenorhabditis elegans N2Caenorhabditis Genetics Center (CGC)Wild type 
CholesterolSigmaC3045
Costa Assay Plate, 96 Well Black With Clear Flat Bottom Non-treated, No Lid PolystyreneCorning IncorporatedREF. 3631
Dimethyl sulfoxideSigmaD2650
Enterococcus faecalis KCTC 3206Korean Collection for Type CultureKCTC NO. 3206Falcutative anaerobic
Escherichia coli OP50Caenorhabditis Genetics Center (CGC)
Fluorescein isothiocyanate - dextranSigmaFD10S
Harmony software PerkinElmerverson 3.5
Luria-Bertani LB mediumMerckVM743185 626  1.10285.5000
Magnesium sulfate heptahydrate Fisher BioreagentsBP2213-1
Fluoromount aqueous mounting mediumSigmaF4680
Operetta CLS High-Content Analysis SystemPerkinElmer HH16000000
PeptoneMerckEMD 1.07213.1000
Pseudomonas aeruginosa PA01Korean Collection for Type CultureKCTC NO. 1637
Sodium ChlorideFisher BioreagentsBP358-1
Stereo MicroscopeNikon, JapanSMZ800N
Yeast extractBecton DickinsonREF. 212750

References

  1. Bischoff, S. C., et al. Intestinal permeability–a new target for disease prevention and therapy. BMC Gastroenterology. 14 (1), 189 (2014).
  2. Peng, L., Li, Z. R., Green, R. S., Holzman, I. R., Lin, J.

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