Name: measuring the effects of bacteria and chemicals on the intestinal permeability of caenorhabditis elegans
Uploaded: 2019-12-03T13:30:00
Description: Watch this Scientific Journal Video about Measuring the Effects of Bacteria and Chemicals on the Intestinal Permeability of Caenorhabditis elegans at JoVE.com

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

1. Preparation of P. aeruginosa PAO1 and Escherichia coli OP50 Culture
<ol>
	<li>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 &#176;C with a shaking speed of 150 rpm.</li>
	<li>Equally distribute the bacterial culture into two 500 mL centrifuge tubes and centrifuge the tubes at 3,220 x g at 4 &#176;C for 30 min.</li>
	<li>Remove the supernatan...

<ol>
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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...

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.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr >
			<td >3,3&#8217;-diindolylmethane&#160;</td>
			<td >Sigma</td>
			<td >D9568</td>
			<td ></td>
		</tr>
		<tr >
			<td >90&#215;15 mm Petri dishes</td>
			<td >SPL Life Sciences, South Korea</td>
			<td >10090</td>
			<td></td>
		</tr>
		<tr >
			<td >60&#215;15 mm Petri dishes</td>
			<td >SPL Life Sciences, South Korea</td>
			<td >10060</td>
			<td></td>
		</tr>
		<tr >
			<td >Bactor Agar</td>
			<td >Beckton Dickinson</td>
			<td >REF. 214010</td>
			<td></td>
		</tr>
		<tr >
			<td >Formaldehyde solution&#160;</td>
			<td >Sigma</td>
			<td >F1635</td>
			<td></td>
		</tr>
		<tr >
			<td >Brain Heart Infusion (BHI)&#160;</td>
			<td >Becton Dickinson</td>
			<td >REF. 237500</td>
			<td></td>
		</tr>
		<tr >
			<td >Caenorhabditis elegans N2</td>
			<td >Caenorhabditis Genetics Center (CGC)</td>
			<td ></td>
			<td>Wild type&#160;</td>
		</tr>
		<tr >
			<td >Cholesterol</td>
			<td >Sigma</td>
			<td >C3045</td>
			<td></td>
		</tr>
		<tr >
			<td >Costa Assay Plate, 96 Well Black With Clear Flat Bottom Non-treated, No Lid Polystyrene</td>
			<td >Corning Incorporated</td>
			<td >REF. 3631</td>
			<td></td>
		</tr>
		<tr >
			<td >Dimethyl sulfoxide</td>
			<td >Sigma</td>
			<td >D2650</td>
			<td></td>
		</tr>
		<tr >
			<td >Enterococcus faecalis KCTC 3206</td>
			<td >Korean Collection for Type Culture</td>
			<td >KCTC NO. 3206</td>
			<td>Falcutative anaerobic</td>
		</tr>
		<tr >
			<td >Escherichia coli OP50</td>
			<td >Caenorhabditis Genetics Center (CGC)</td>
			<td ></td>
			<td></td>
		</tr>
		<tr >
			<td >Fluorescein isothiocyanate - dextran</td>
			<td >Sigma</td>
			<td >FD10S</td>
			<td></td>
		</tr>
		<tr >
			<td >Harmony software&#160;</td>
			<td >PerkinElmer</td>
			<td ></td>
			<td>verson 3.5</td>
		</tr>
		<tr >
			<td >Luria-Bertani LB medium</td>
			<td >Merck</td>
			<td >VM743185 626&#160; 1.10285.5000</td>
			<td></td>
		</tr>
		<tr >
			<td >Magnesium sulfate heptahydrate&#160;</td>
			<td >Fisher Bioreagents</td>
			<td >BP2213-1</td>
			<td></td>
		</tr>
		<tr >
			<td >Fluoromount aqueous mounting medium</td>
			<td >Sigma</td>
			<td >F4680</td>
			<td></td>
		</tr>
		<tr >
			<td >Operetta CLS High-Content Analysis System</td>
			<td >PerkinElmer</td>
			<td>&#160;HH16000000</td>
			<td></td>
		</tr>
		<tr >
			<td >Peptone</td>
			<td >Merck</td>
			<td >EMD 1.07213.1000</td>
			<td></td>
		</tr>
		<tr >
			<td >Pseudomonas aeruginosa PA01</td>
			<td >Korean Collection for Type Culture</td>
			<td >KCTC NO. 1637</td>
			<td></td>
		</tr>
		<tr >
			<td >Sodium Chloride</td>
			<td >Fisher Bioreagents</td>
			<td >BP358-1</td>
			<td></td>
		</tr>
		<tr >
			<td >Stereo Microscope</td>
			<td >Nikon, Japan</td>
			<td >SMZ800N</td>
			<td></td>
		</tr>
		<tr >
			<td >Yeast extract</td>
			<td >Becton Dickinson</td>
			<td >REF. 212750</td>
			<td></td>
		</tr>
	</tbody>
</table>

measuring the effects of bacteria and chemicals on the intestinal permeability of caenorhabditis elegans

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.

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 &#177; 6.6, 369.7 &#177; 38.9, and 105.6 &#177; 10.6%, respectively. The data emphasize that P. aeru...

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Measuring the Effects of Bacteria and Chemicals on the Intestinal Permeability of Caenorhabditis elegans

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.

Measuring the Effects of Bacteria and Chemicals on the Intestinal Permeability of Caenorhabditis elegans

In living organisms, intestinal hyperpermeability is a serious symptom that leads to many inflammatory bowel diseases (IBDs). Caenorhabditis elegans is a ...

This method will be helpful for basic biological studies related to the interaction between intestinal microbiota and gut health. In addition, this protocol may be used for screening potential probiotics and nutraceuticals to cure intestinal health problems, such as leaky gut syndrome and inflammatory bowel diseases. This protocol uses a high-throughput imaging system to enable rapid and precise quantitative analysis of intestinal permeability in C.elegans treated with various bacteria and chemicals.We recommend that a scientist who is not familiar with this technique limits the number of samples in order to avoid mislabeling and other mistakes. This procedure consists of many steps that require visual demonstration, such as transferring worms to 96-well plates and using the Operetta machine, as well as the Harmony software. Start by preparing 500 milliliters of sterile LB medium.Inoculate a single colony of P.aeruginosa into the medium, and incubate the culture for 14 to 15 hours at 37 degrees Celsius while shaking at 150 rpm. The next day, evenly distribute the bacterial culture into two 500-milliliter tubes, and centrifuge them at 3, 220 times g and four degrees Celsius for 30 minutes. Remove the supernatant, leaving 25 milliliters in each tube, for a total remaining volume of 50 milliliters, then resuspend the bacterial pellet.Store the bacterial culture at four degrees Celsius until ready to use. To test the effects of bacteria on the intestinal permeability of C.elegans, remove the cultures from the refrigerator, and thoroughly vortex them. Add a total of 800 microliters to each fresh NGM plate, and allow the plates to dry in a 20-degree Celsius incubator overnight.To prepare DIM plates, add 50 milliliters of NGM agar medium into a bottle of DIM containing NGM broth, and mix thoroughly. Then, pour 20 milliliters of the medium into each 90-by-15-millimeter Petri dish. Prepare DMSO plates by adding 20 milliliters of DMSO containing NGM medium to each Petri dish, and leave the plates at room temperature for at least three hours to solidify.Spread 800 microliters of vortexed E.coli or P.aeruginosa culture onto each fresh NGM plate, and allow the plates to dry in a 20-degree Celsius incubator overnight. Wash the age-synchronized L4 larvae with S-buffer, and transfer approximately 500 worms to the NGM plates containing different bacteria and chemicals. Then, incubate the plates at 20 degrees Celsius for 48 hours.Prepare the FITC-dextran-supplemented plates by mixing two milliliters of heat-inactivated E.coli with four milligrams of FITC-dextran. Add 100 microliters of the mixture to each of 20 fresh NGM agar plates, and allow the plates to dry for one hour on a clean lab bench. After 48 hours of treatment, wash the worms with S-buffer, transfer them to FITC-dextran-supplemented plates and to NGM plates without FITC-dextran, and incubate the plates overnight.The next day, wash the worms with S-buffer, and allow them to crawl in the fresh NGM agar plate for one hour. Add 50 microliters of 4%formaldehyde to each well of a black, 96-well, flat-bottomed plate, and transfer approximately 50 worms into each well. After one to two minutes, remove all the formaldehyde, and add 100 microliters of mounting medium into each well.Formaldehyde is a toxic chemical and should be handled with care. To capture fluorescent images, press the Open Lid icon within the imaging software, and insert the plate into the machine. Click Setup, select the plate type, and add the brightfield and EGFP channels.To adjust the layout, go to the Layout selection, and select Track. Set the first picture at one micrometer, number of planes to 10, and the distance to one micrometer. Select one of the treatment wells and one capture field, and press Test in the Run Experiment section.If the images are satisfactory, return to the Setup section, and press the Reset icon. Then, select all the target wells and a suitable number of capture fields. Go to Run Experiment, enter the plate name, and press Start.To measure fluorescence intensity, go to the Image Analysis section, input the image, and find the cell by choosing the EGFP channel and method B.Adjust the parameters according to the manuscript directions, and choose the mean as the output. Press the Apply icon to save the setup. Obtain a heat map and a data table by going to the Evaluation section and adjust the readout parameter, then start the evaluation.C.elegans showed a significant increase in FITC-dextran fluorescence after incubation with P.aeruginosa compared with the other two bacterial strains, indicating that P.aeruginosa caused more vital damage to the epithelial gut barrier and increased intestinal permeability. Live and heat-inactivated P.aeruginosa triggered very different effects on the intestinal permeability in C.elegans. Upon heat inactivation, both the fluorescence images and statistical data indicate that P.aeruginosa did not trigger any toxicity to nematodes.A 48-hour co-treatment with DIM significantly decreased the FITC-dextran fluorescence intensity inside the guts of worms exposed to P.aeruginosa, which demonstrates that DIM can be considered a good natural product to cure intestinal permeability dysfunction caused by bacterial infections. It's important to remember that during the preparation of the FITC-dextran-coated plates, you need to minimize the light exposure because of the light sensitivity of FITC-dextran. This technique can be used to determine the pathogenic and probiotic effects of intestinal bacteria and their active components by testing the bacteria, as well as their chemical composition.

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