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In This Article

  • Summary
  • Abstract
  • Introduction
  • Protocol
  • Results
  • Discussion
  • Disclosures
  • 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 staining.

There are two representative protocols for measuring the gut permeability in C. elegans using Nile red3 and erioglaucine disodium (or the Smurf assay)4,5. In this protocol, we used FITC-dextran (average molecular weight 10,000), which has a much higher molecular weight than Nile red (MW = 318.37) and erioglaucine disodium (MW = 792.85). FITC-dextran is more similar than Nile red or erioglaucine disodium dyes to actual macromolecular nutrients such as carbohydrates, which are absorbed through the intestinal layer. The intestinal permeability of C. elegans fed with erioglaucine disodium (blue Smurf dye) can be easily evaluated without fluorescence microscopy. However, in the Smurf assay, quantitative analysis of intestinal permeability is difficult due to the lack of standardization and should be evaluated manually4,5. In the case of the Nile red assay, Nile red also stains lipid droplets in cells, which may interfere with the exact determination of gut permeability in C. elegans6. The present protocols enable rapid and precise quantitative analysis of intestinal permeability in C. elegans treated with various intestinal bacteria and chemicals while avoiding unspecific lipid staining.

C. elegans is a typical model in biological fields due to its affordable price, easy manipulation, limited animal ethics issues, and short lifespan, which is beneficial for rapid experimentation7. In particular, after the entire C. elegans genome was published, nearly 40% of genes in the C. elegans genome were found to be orthologous to genes that cause human diseases8. Moreover, the transparent body allows observation inside the organism, which is advantageous for researching cellular events and for fluorescence applications in cell biology, for example, stem cell staining with DAPI or immunohistochemistry9. C. elegans is often used as an experimental animal to study the interaction between the gut microbiota and the host; in addition, C. elegans is used to screen health-promoting probiotic bacteria10,11,12 as well as dietary chemicals promoting intestinal health13,14.

Pseudomonas aeruginosa and Enterococcus faecalis are well-known gut bacteria that negatively affect the gastrointestinal system, especially the colonic epithelial cells of the intestinal tract15,16. Therefore, measuring the gut permeability triggered by these bacteria is necessary for the screening and development of new drugs that can recover and reduce the damage caused by bacterial inflammation and infection. In this protocol, we tested the effects of these intestinal bacteria on the intestinal permeability of C. elegans.

We also report an optimized protocol for testing chemicals on the intestinal permeability of C. elegans. For this purpose, we used 3,3'-diindolylmethane (DIM) as a model chemical because DIM is a bioactive metabolite compound derived from indole-3-carbinol, which is present in Brassica food plants, and has been reported to have therapeutic effects on IBD in mice17,18. In addition, we recently discovered that DIM improves intestinal permeability dysfunction in both cultured human intestinal cells as well as the model nematode C. elegans19.

In this study, we used three different experimental conditions. First, we measured the effects of the different bacteria, P. aeruginosa and E. faecalis, on intestinal permeability (Figure 1). Second, we measured the effects of live and heat-inactivated P. aeruginosa on intestinal permeability (Figure 2). Third, we measured the effects of DIM (a model chemical) on the intestinal permeability of C. elegans fed with P. aeruginosa (Figure 3).

The objective of this study was to develop optimized protocols that measure the intestinal permeability of C. elegans, which is changed by treatment with various intestinal bacteria as well as with chemicals.

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 supernatant until the volume is 50 mL (one-tenth of the initial volume) and resuspend the bacterial pellet.
  4. Store the concentrated bacterial culture at 4 °C until use. The storage period of P. aeruginosa culture can be up to 1 month, but fresh culture is better for inducing intestinal damage.
    NOTE: The protocol can be paused here. In addition to the whole live bacteria, the supernatant of the bacterial culture can be used to test the effects of the intestinal bacteria19.
  5. To prepare E. coli OP50, culture E. coli OP50 in DYT medium (Table 1) and then apply similar steps to those described above but decrease the volume to one-sixth of the original volume. For example, if the initial volume is 500 mL, after removing supernatant, the remaining volume is approximately 83 mL.
    NOTE: The protocol can be paused here.

2. Preparation of Enterococcus faecalis KCTC 3206 Culture

  1. Prepare 500 mL of sterilized brain heart infusion (BHI) broth (Table 1).
  2. Inoculate one colony into 500 mL of BHI broth and incubate the culture for 14–15 h in a shaking incubator at 37 °C and 150 rpm.
  3. 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.
  4. Remove the supernatant until the volume is 50 mL (one-tenth of the initial volume) and resuspend the pellet.
  5. Store the concentrated bacterial culture at 4 °C until use. The fresh culture is better for testing the effects on the intestinal permeability.
    NOTE: The protocol can be paused here.

3. Preparation of Heat-inactivated E. coli OP50 and Heat-inactivated P. aeruginosa PAO1 Cultures

  1. Culture and harvest the bacteria as described above (steps 1.1–1.3).
  2. Heat-inactivate the E. coli OP50 or P. aeruginosa PAO1 culture as described previously20,21,22. For heat inactivation, incubate the resuspended bacteria at 65 °C (water bath) for 30 min.
  3. Cool the concentrated bacterial culture to room temperature and store it at 4 °C until use. The storage period can be up to 1 month.
    NOTE: The protocol can be paused here.

4. Preparation of Nematode Growth Medium (NGM) Plates for Testing the Effects of Different Bacteria on the Intestinal Permeability of C. elegans

  1. Place 1.25 g of peptone, 1.5 g of NaCl, 8.5 g of agar, a magnetic stirrer and 487.5 mL of distilled water in a 500 mL glass bottle (Table 1).
  2. Mix the mixture well, autoclave the mixture for 15 min at 121 °C and cool the mixture to 55 °C in a water bath for 30 min.
  3. Remove the medium from the water bath, add the components (0.5 mL of 1 M CaCl2, 0.5 mL of cholesterol, 0.5 mL of MgSO4, 12.5 mL of KPO4) (Table 1) to the NGM, and mix well.
    NOTE: All the individual components must be autoclaved except for cholesterol (dissolved in absolute ethanol), and every experimental step must be carried out on a clean bench.
  4. Distribute 20 mL of NGM to each 90 x 15 mm Petri dish and allow the agar to solidify on a clean bench at room temperature (approximately 20 °C). The NGM plates can be stored for up to one month at 4 °C.
    NOTE: The protocol can be paused here. The NGM plates used for FITC-dextran staining were prepared by the same procedure (from steps 4.1–4.3). 10 mL of NGM was distributed to each 60 x 15 mm Petri dish and allowed to solidify on a clean bench at room temperature (approximately 20 °C).
  5. Remove the bacterial culture from the 4 °C refrigerator and vortex the culture thoroughly before spreading the culture onto the NGM plates.
  6. Add a total of 800 µL of the bacterial culture to each fresh NGM plate, and allow the plates to dry in a 20 °C incubator overnight.
    NOTE: For the first experiment (Figure 1), two NGM plates with E. coli OP50, one NGM plate with P. aeruginosa PAO1, and one NGM plate with E. faecalis KCTC3206 were prepared. For the second experiment (Figure 2), two NGM plates with live E. coli OP50, one NGM plate with live P. aeruginosa PAO1, and one NGM plate with heat-inactivated P. aeruginosa PAO1 were prepared.

5. Preparation of NGM Plates for Testing the Effects of a Chemical (DIM) on the Intestinal Permeability of C. elegans Fed with P. aeruginosa

  1. Add 0.5 g of peptone, 0.6 g of NaCl, 195 mL of distilled water, a magnetic stirrer, and 6.8 g of agar to a 500 mL glass bottle (NGM agar).
  2. Add 0.5 g of peptone, 0.6 g of NaCl, 195 mL of distilled water, and a magnetic stirrer without agar to another 500 mL glass bottle (NGM broth).
  3. Autoclave the two bottles (from steps 5.1–5.2), one empty 100 mL glass bottle, and one empty 500 mL glass bottle for 15 min at 121 °C. Then, allow the medium containing bottles to cool to 55 °C in the water bath for 30 min. Keep the agar medium in the water bath and remove the bottle containing broth (from step 5.2) for the next step.
  4. Add the additive chemicals to the NGM broth: 0.4 mL of 1 M CaCl2, 0.4 mL of cholesterol in ethanol (mL), 0.4 mL of 1 M MgSO4 and 10 mL of 1 M KPO4 (all of these components must be sterilized apart from the cholesterol in ethanol). Then, stir the mixture thoroughly with a magnetic stirrer at 55 °C.
  5. Label the autoclaved empty 500 mL glass bottle with dimethyl sulfoxide (DMSO) and the autoclaved empty 100 mL glass bottle with DIM.
  6. Transfer 50 mL of NGM broth into the DIM-labeled 100 mL bottle. Add 500 µL of 20 mM DIM stock into the bottle and mix well.
    NOTE: DIM is dissolved in DMSO (20 mM DIM stock).
  7. Quickly remove the NGM agar medium from the water bath, add 50 mL of the NGM agar medium into the DIM-labeled bottle and mix thoroughly.
  8. Place a 20 mL aliquot of DIM-containing NGM medium into each 90 mm x 15 mm Petri dish. Approximately five DIM-containing NGM plates can be made from this step.
    NOTE: The final concentration of DIM in each NGM plate will be 100 µM.
  9. To prepare the DMSO-containing NGM plate, transfer 150 mL of NGM broth into the DMSO-labeled 500 mL bottle. Add 1.5 mL of DMSO to the bottle and mix well.
  10. Add 150 mL of the NGM agar medium to the DMSO-labeled bottle and mix thoroughly.
  11. Place a 20 mL aliquot of DMSO-containing NGM medium in each 90 mm x 15 mm Petri dish. Approximately fifteen DMSO-containing NGM plates can be made from this step.
    NOTE: The final concentration of DMSO in each NGM plate will be 0.5%.
  12. Solidify the plates at room temperature for at least 3 h and store them at 4 °C until use.
    NOTE: The protocol can be paused here.
  13. Remove the E. coli OP50 or P. aeruginosa PAO1 culture from the 4 °C refrigerator (from step 1.4) and vortex the culture properly before spreading onto the NGM plates.
  14. Put 800 µL of the E. coli OP50 or P. aeruginosa PAO1 bacterial culture onto each fresh NGM agar plate and allow the plates to dry in a 20 °C incubator overnight.
    NOTE: The protocol can be paused here. For the third experiment (Figure 3), two DMSO-containing NGM plates coated with live E. coli OP50, one DMSO-containing NGM plate coated with live P. aeruginosa PAO1, and one DIM-containing NGM plate coated with live P. aeruginosa PAO1 were prepared.

6. Preparation of Age-synchronized C. elegans

  1. Grow worms on a solid NGM plate and feed them with E. coli OP50 until the desired population is reached.
  2. Synchronize the eggs by using either the timed egg laying method or the bleaching solution treatment as described previously20,23,24.

7. Treatment of Bacteria or DIM and FITC-dextran Feeding

  1. Incubate the age-synchronized eggs at 20 °C for 64 h on NGM plates supplemented with live E. coli OP50 as food.
  2. Wash the age-synchronized L4 larva with S-buffer and transfer them (more than approximately 500 worms) to the treatment NGM plates containing different bacteria and chemicals (described in NOTE following step 4.6 and 5.14,). Incubate them at 20 °C for 48 h.
    NOTE: The treatment time can range from 24 to 72 h, according to the bacteria and chemicals used. The therapeutic or preventive effect of DIM can also be evaluated by pretreating the worms with pathogens and then treating the worms with DIM (the therapeutic effect) or by pretreating the worms with DIM and then treating with pathogens (the preventive effect)19.
  3. To prepare the FITC-dextran-supplemented plates, mix 2 mL of heat-inactivated E. coli OP50 with 4 mg of FITC-dextran. Then, divide 100 µL of the FITC-dextran and E. coli OP50 mixture into twenty fresh NGM agar plates (60 mm x 15 mm) and allow the plates to dry for 1 h on a clean bench.
    NOTE: The final concentration of FITC-dextran in each NGM plate will be 20 μg/mL.
  4. Prepare five E. coli OP50-containing NGM plates without FITC-dextran for the vehicle control treatment. For this purpose, divide 100 µL heat-inactivated E. coli OP50 to each fresh NGM agar plates (60 mm x 15 mm) and allow the plates to dry for 1 h on a clean bench.
    NOTE: For each independent experiment, fifteen FITC-dextran-supplemented NGM plates and five NGM plates without FITC-dextran are required.
  5. After 48 h of treatment (from step 7.2), wash the worms with S-buffer, transfer the worms to the FITC-dextran supplemented plates and the NGM plates without FITC-dextran, and incubate the plates overnight (14–15 h).
    NOTE: For each treatment group, 5 replicates of FITC-dextran staining (or vehicle control feeding) are required.
  6. Wash the worms with S-buffer and allow them to crawl in the fresh NGM agar plate for 1 h.
    NOTE: For each independent experiment, a total of 20 fresh NGM plates are needed. In this step, you can use the NGM plate supplemented without or with E. coli OP50.
  7. Add 50 µL of 4% formaldehyde solution to each well of a black 96-well flat-bottom plate. Transfer approximately 50 worms from each NGM plate into each well for fluorescence measurements. After 1 to 2 min, thoroughly remove all the formaldehyde from each well and add 100 µL of mounting medium to coat the wells.
    NOTE: Formaldehyde solution (4%) is used to immobilize and fix the worms. For each treatment group, 5 wells (5 replicates) are used for image analysis.

8. Imaging C. elegans with the Operetta Imaging System and Determination of Intestinal Permeability by Measuring the FITC-dextran Fluorescence Uptake

NOTE: Fluorescent stereomicroscopy can be used for image analysis instead of the Operetta system.

  1. Capture fluorescence images and measure the fluorescence intensity using the Operetta High-Content Imaging System and analyze the images with Harmony software.
  2. In the Harmony software, press the icon Open lid to open the lid and to put the plate into the machine.
  3. Set up the parameters.
  4. Click Set up, select the plate type (96-well corning flat-bottom) and add the channels (bright-field and EGFP channels).
  5. Adjust the layout. Go to the Layout selection, and then select Track and adjust the parameters (first picture at 1 µm, number of planes are 10, distance is 1 µm).
  6. Select one of the treatment wells and one capture field and press Test to check whether the pictures are satisfactory in the Run experiment section.
  7. If the pictures are satisfactory, return to the Set up section and press the Reset icon at the end of the screen. Then, select all the target wells and a suitable number of capture fields.
  8. Go to Run experiment again and enter the plate name, then press Start to begin processing.
  9. To measure the intensity of the fluorescence, go to the image analysis section and input the image. Find the cell by choosing the EGFP channel and method B. Adjust the common threshold to 0.5, area to >200 µm2, split factor to 3.0, individual threshold to 0.18 and contrast to more than 0.18. Then, calculate the intensity properties and choose the mean as the output. Press the Apply icon to save the setup.
  10. Go to the Evaluation section to measure the intensity by obtaining a heatmap and data table. Set the Readout parameter to Cells — Intensity Cell EGFP Mean — Mean per Well and start the evaluation.
    NOTE: The protocol can be paused here.
  11. To extract the data from Operetta, click the Setting button and choose Data management.
  12. Choose Write archive, and then open the browse and select the file.
  13. To select the file, click the small + signal at the left corner, and then click Measurement and choose Plate name.
  14. Select the file and click OK.
  15. Select the path to save the file by clicking the Browse signal at the active path.
  16. Click Start to save the data file.
    NOTE: The protocol can be paused here.

9. Statistical Analysis of the FITC-dextran Fluorescence of C. elegans

  1. Import the data and calculate the mean and standard deviation (SD) using a statistics software.
  2. Analyze the significant difference with one-way analysis of variance (ANOVA) followed by Tukey's multiple comparison test.

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...

Disclosures

The authors have nothing to disclose.

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

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