assessing intestinal permeability in caenorhabditis elegans using smurf staining

Evaluating Metabolism and Aging Pattern in &lt;i&gt;Caenorhabditis elegans&lt;/i&gt;

Biological sciences teaching in high school is a continuous challenge. Notably, the access and use of technology have brought important advances in the teaching-learning process, however, tools such as artificial intelligence chatbots make rationalizing and seeking evidence more difficult due to easy (and sometimes incorrect) responses obtained1. Because of that, the use of a scientific method with practical experimentation in an inquiry-based approach in the classroom is an important strategy to develop or stimulate critical thinking, creativity, and technical skills in the students2.
In this context, the free living nematode Caenorhabditis elegans has been successfully used in experimentation for teaching purposes3 because of its particular advantages: It is not a parasite and the Escherichia coli used for feeding is biosafety level-1, therefore reducing near to zero the biological hazard; it has an elegant and quantifiable locomotion movement, which is interesting for students to observe; and it is transparent, which allows organ observation, but also staining with pigments that can indicate the presence of biomolecules or the occurrence of physiological alterations4. Therefore, it is possible to hypothesize and test in the classroom simple postulations related to biochemistry and physiological changes such as aging.
Glycogen is a storage carbohydrate, formed by a long and branched chain of glucose molecules formed by glucosyl residues with (1&#8594;4)-&#945; glycosidic linear linkages and (1&#8594;6)-&#945; glycosidic bonds at branch points and is particularly important for muscle contraction, cell differentiation and glycemia maintenance5. Glycogen is synthesized after feeding due to insulin activation of the enzyme glycogen-synthase. During exercise or fasting, epinephrine or glucagon, respectively, activate glycogen phosphorylase and, therefore, break down the polysaccharide to provide glucose-6-phosphate to the muscle cells or release free glucose to circumvent hypoglycemia6,7. Alterations in glycogen levels impacts cell differentiation, signaling, redox regulation, and stemness under various physiological and pathophysiological conditions, including cancer8. In C. elegans, glycogen is mainly found in esophageal muscle, hypodermis, intestine, neurons and mainly in body wall muscles9. The glycogen content can be measured by using Lugol&#39;s Iodine solution, since iodine binds into the helical coils forming an iodine-glycogen complex, giving a visible sharp blue-black or brown-black color, which has been successfully used to demonstrate glycogen content in C. elegans10. It has been demonstrated that glycogen accumulation caused by high glucose feeding can reduce worm&#39;s lifespan, therefore accelerating the aging process11,12. In addition, metabolic disturbances, other hormones, and exposure to xenobiotics can alter glycogen metabolism as well13,14. Therefore, experimentation on glycogen content in C. elegans is quite interesting, since diverse factors may disturb its metabolism and can stimulate an in-class discussion on basic biochemistry associated with transversal themes such as exercise, diets, diseases, and aging.
Aging is a time-dependent functional decline caused by cellular damage. This damage can be associated with oxidative stress, telomere attrition, loss of proteostasis, inflammation and even by accumulation of insoluble polyglucosan bodies15, just to name a few. One of the hallmarks of aging is the reduction of intestinal integrity, associated with several chronic conditions that occur during the life of an organism16. Maintenance of intestinal homeostasis depends on the integrity of the intestinal epithelium, which is supported by junctional proteins forming a physical barrier and connecting adjacent epithelial cells. When there is damage to this epithelium, leakage of luminal content into the interstitium occurs17. Based on this mechanism, the smurf test has been used to verify intestinal integrity in several animal models, since this blue dye Erioglaucine disodium salt does not cross the intestinal membrane, remaining in the lumen18. When worms are infected with a pathogen, contaminated with some toxicants or age, altering the interstitial integrity, the dye crosses the barrier and spreads all over the worm, which becomes all blue. This assay allows discussion on the physiology of aging and experimenting on factors that can accelerate or delay this process by exposing worms to different conditions. The protocols here will describe in detail these two, dye-based methods that can be easily done in class to instigate and stimulate students to formulate and test hypotheses related to biochemistry and physiology.
The first part of protocol shows its applicability to analyze qualitatively and quantitatively the glycogen content in C. elegans model10. The purpose of second part of the protocol is to assess the integrity of the C. elegans intestine. This technique allows for the monitoring of C. elegans aging by evaluating the integrity of the intestinal membranes. Furthermore, it allows evaluating whether a substance accelerates or delays aging and whether any substances have toxic potential on the intestinal barrier19.
The C. elegans strain used for the present study was Bristol N2 wild type. However, the procedure can be replicated using strains that exhibit comparable growth rates, or the method must be adjusted based on need of equipment replacement, considering they have the same or similar function, or depending on the strain used, as certain strains have specific maintenance and/or sensitivity requirements; this information can be obtained from the Caenorhabditis Genetics Center (CGC) or WormBase website. These changes should not impact the reproducibility of the method.

Author Spotlight: Exploring Metabolic and Aging Processes in &lt;i&gt;C. elegans&lt;/i&gt;  Using Low-Cost, High-Impact Assays

Using Low-Cost Dyes to Visualize Glycogen Accumulation and Gut Integrity in Caenorhabditis elegans

The nematodes in Caenorhabditis elegans is easy to cultivate, to maintain, to manipulate, and to be used in experiments for biological science teaching, and also for students to carry out their projects. This protocol brings two easy and low cost assays that are based on the transparency of the nematode, and also on on the use of colored dyes. The first protocol allows the investigation of metabolic chains following different diets, and the second protocol allows the observation of the aging process in a short period of time.These both assays are interesting for educational purposes, under limited resources. The first assay takes an advantage of the worm's transparency, and the fact that it produce and it stores glycogen. Using the Lugol iodine solution, we can dye the worms, and verify whether different diets or other factors can modify its levels.Aging is a natural process, but some factors such as ultraviolet, food and chemicals exposure can accelerate this process. The intestinal barrier is modified with the agent, and the epithelial loses integrity, causing leakage of the substance the worms intake, such as the walls thin. We call this assay Smurf test, because the worms get totally blue when there is a reduction of intestinal integrity.

Assessing Intestinal Permeability in Caenorhabditis elegans Using Smurf Staining

To begin, transfer 500 to 1, 000 L1 stage synchronized Caenorhabditis elegans worms on NGM agar plate and incubate at 20 degrees Celsius until the day of the test. Design the assay schedule for two experimental groups. Using M9 buffer, collect L4 worms and adulthood worms on the respective day.Transfer the collected worms to 1.5 milliliter micro tubes and wait for worms to sediment by gravity. Wash the worms thrice with one milliliter of M9 buffer, leaving approximately 500 microliters in the tube after the last wash. Using an automatic pipette, add 10 microliters of the worm suspension to a microscope slide and calculate the volume required to obtain 100 worms per microliter.In a new micro tube, add the volume required for 100 worms and 100 microliters of 25%erioglaucine disodium salt solution. Then, add 200 microliters of E.coli OP50 culture and increase the volume to 500 microliters using an M9 buffer. Incubate the tube for three hours with agitation in a mixer, protecting it from light.Upon incubation, allow the worms to sediment by gravity before washing them with one milliliter of M9 buffer until the staining solution is completely removed. After the last wash, resuspend the worms in 250 microliters of M9 buffer and transfer approximately 50 microliters onto the slide. Place the cover slip and incubate the slide at 20 degrees Celsius for 10 minutes to paralyze the worms.Using a stereo microscope, under Bright Field mode, count the total number of worms and the total dyed worms. Intestinal permeability assay displayed a strong intestinal barrier in young worms preventing dye leakage. In contrast, aged worms exhibited dye extravasation throughout their bodies, indicating intestinal membrane damage.

assessing-intestinal-permeability-caenorhabditis-elegans-using-smurf

Research

JoVE Journal

Biology

Caenorhabditis elegans (C. elegans) is a transparent, non-parasitic nematode with a simple biology, which makes it a great tool for biological sciences teaching through the staining of the cells or their molecular content. Lugol dye (iodine-potassium iodide solution) has been widely used in biochemistry to stain glycogen stores. In this context, it is possible to observe differences between fed and starved animals, besides the effects of different conditions, such as different diets and oxygen levels. Erioglaucine is a blue dye that indicates the loss of the intestinal barrier. When the intestinal barrier is intact, the blue dye stains inside the lumen; however, when this integrity is disrupted, the dye leaks into the body cavity. Using a stereomicroscope or a microscope, teachers can demonstrate physiological and biochemical alterations, or they can instigate students to ask a scientific question and hypothesize and test their hypothesis using these assays. The present protocol describes two staining techniques in C. elegans that can be easily carried out by students.

Teaching biological sciences can be made more stimulating for students through the use of experimentation. This manuscript presents two different yet complementary protocols that can be utilized in the classroom to encourage students to formulate and test hypotheses related to high-calorie diets, starvation, and aging.

Caenorhabditis elegans (C. elegans) is a transparent, non-parasitic nematode with a simple biology, which makes it a great tool for biological sciences ...

Assessing Glycogen Content in Caenorhabditis elegans Using Lugol&#39;s Iodine Staining

To begin, prepare six nematode growth media or NGM agar plates. The next day, add 200 microliters of Escherichia coli OP50 culture onto the plates, and incubate at room temperature for one day. After incubation, add 200 microliters of 0.025 molar D-glucose to four NGM agar plates, and allow them to dry at room temperature.Transfer different staged three to four chunks of Caenorhabditis elegans Bristol N2 from a maintenance NGM agar plate to a new NGM plate seated with E.coli OP50. Incubate the C.elegans worms at 20 degrees Celsius with humidity of more than 95%for three days. Three days after, add distilled water onto the plate, and using a Pasteur pipette, collect the worms.Transfer the collected worms to a 50 milliliter centrifuge tube, and allow them to sediment by gravity before removing the supernatant from the tube. Then, add distilled water into the tube. Once the volume of the tube is reduced to five milliliters, add 10 milliliters of bleaching solution and vigorously shake the tube for approximately six minutes.Then, fill the tube to a 50 milliliter capacity with M9 buffer, and centrifuge at 1400 g for three minutes. Remove the supernatant up to five milliliters and add 45 milliliters of M9 buffer into the tube. After the last wash, remove the supernatant up to the 15 milliliter mark and transfer the remaining liquid to a plate.Observe the plate under the microscope for the release of eggs, and place the plate at 20 degrees Celsius with humidity of more than 95%for 14 hours. After incubation, collect the worms into a 15 milliliter tube. Using an automatic pipette, add 10 microliters of the synchronized worms to a microscope slide.Count the number of worms and calculate the volume required to obtain 500 to 1000 worms per microliter. Transfer 500 to 1000 larval stage one, or L1, synchronized worms to the NGM agar plates. Previously prepared and seeded with E.coli OP50 and glucose.Incubate the plate at 20 degrees Celsius until the worms reach larval stage four, or L4.After 48 hours, add M9 buffer onto the plate, and using a Pasteur pipette, collect L4 larvae. Transfer the collected larvae to a 1.5 milliliter tube. Design the assay schedule for three experimental groups.Transfer approximately 100 microliters of worm suspension from each group to the 1.5 milliliter microtube containing 400 microliters of 5%Lugol's Iodine Solution. Gently agitate the tube in a mixer for five minutes. After removing the tube from the mixer, wait for worms to sediment by gravity.Wash the worms thrice with one milliliter of M9 buffer. After re-suspending the worms in 100 microliters of M9 buffer. Transfer approximately 50 microliters onto the slide.Place the cover slip onto the slide. Next, on a stereo microscope, select the bright-field mode and observe the worms. Save the images containing a minimum of 10 worms per group as a jpeg file.Finally, calculate the glycogen content based on the iodine staining of worms. Visual observations of glycogen content assay revealed that fasting worms displayed a weaker stain compared to those fed with E.coli OP50 and those with E.coli OP50 and D-glucose, which exhibited the most intense staining.