Effects of Polyphenols on Motor Movement Improvement in Parkinson’s Modeled C. elegans

Table: MED1219
Experimentation location: School
Regulated Research (Form 1c): No
Project continuation (Form 7): No

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Abstract:

The neurodegenerative disorder, Parkinson’s disease (PD), is a debilitating disease that affects millions worldwide. Due to the complexity of the disorder, the etiology is still unknown. It is understood that PD degenerates dopamine neurons in the substantia nigra, effectively impairing motor movement. Caenorhabditis elegans is a free-living nematode that is a model organism due to its simplicity and shared molecular pathways and genomes with humans. Because of its simplicity, mutant forms can be used to study neurological diseases and pathological conditions, including PD. A variety of internal and external factors are speculated to be involved in the rate of degeneration of said dopaminergic neurons in the substantia nigra concurrently with PD. Nutrition intake is one of the external factors said to play a role in neuroprotection in neurological disorders such as PD. Polyphenols, of the phenol group of naturally occurring organic chemical compounds, are found in many superfoods and are presumed to have a positive impact on neurodegenerative disease.
In this experiment, I focused on the use of a genetically modified strain of C. elegans and tested the motor movement improvement using a chemotaxis assay and calculated the chemotaxis index of each plate. After calculating the chemotaxis index, it was determined that nutrition and the addition of polyphenols could potentially hold some significance in the motor movement improvement of C. elegans and, by extension, PD in humans. This approach could provide a less allopathic pathway to prevent and treat neurological conditions. Combined with allopathic medicine, the use of polyphenols in treatment could potentially bolster neuroprotection and provide alleviation to those battling PD.

Bibliography/Citations:

 

Abdullah R;Basak I;Patil KS;Alves G;Larsen JP;Møller SG;. (n.d.). Parkinson's disease and age: The obvious but largely unexplored link. Retrieved March 08, 2021, from https://pubmed.ncbi.nlm.nih.gov/25261764/

Libretexts. (2021, January 03). 1.19: Pouring agar plates. Retrieved March 08, 2021, from https://bio.libretexts.org/Bookshelves/Biotechnology/Lab_Manual%3A_Introduction_to_Biotechnology/01%3A_Techniques/1.19%3A_Pouring_Agar_Plates

Margie, O., Palmer, C., & Chin-Sang, I. (2013, April 27). C. elegans chemotaxis assay. Retrieved March 08, 2021, from https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3667641/

Parkinson's: Neurons destroyed by three simultaneous strikes. (2009, May 04). Retrieved March 08, 2021, from https://www.sciencedaily.com/releases/2009/04/090429132222.htm#:~:text=The%20studies%20found%20that%20neurons,in%20the%20cell%20over%20time.

Poulose, S., Miller, M., Scott, T., & Shukitt-Hale, B. (2017, November 15). Nutritional factors affecting adult neurogenesis and cognitive function. Retrieved March 08, 2021, from https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5683005/

Uttara, B., Singh, A., Zamboni, P., & Mahajan, R. (2009, March). Oxidative stress and neurodegenerative diseases: A review of upstream and downstream antioxidant therapeutic options. Retrieved March 08, 2021, from https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2724665/

W;, N. (n.d.). Cell death of dopamine neurons in aging and parkinson's disease. Retrieved March 08, 2021, from https://pubmed.ncbi.nlm.nih.gov/10656535/

 


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Research Plan:

Plating

  1. Place the NGM agar with the lid askew into a pot of cold water. 

  2. Heat the water while the bottle of NGM agar is in the pot; this will prevent the glass bottle from cracking due to sudden heat. 

  3. Wait 30 minutes and then return to observe whether the agar has melted completely.

  4. If the agar has melted completely, take it off of the heat and leave it to cool for 10-15 minutes. 

  5. Uncap the media bottle and hold the bottle in your dominant hand. Note: once the bottle is opened, do not talk. Talking will allow bacteria from your mouth to become airborne and may contaminate the media.

  1. The cap can be held in the same hand (between fingers) as your bottle or can be placed on a disinfected surface.

  2. Grab a plate with your other hand and slide it towards the edge of the table, while keeping it closed.

  3. Once the plate is at the edge, open the lid as if there is an imaginary hinge at one end; so the plate opens like a clamshell.

  4. Pour the media into the bottom of the plate until it just covers the surface. Do not over fill.

  5. Close the lid and allow to cool. The media will be solid.

  6. Leave the plates out for a day if possible so the condensation will evaporate from the plate. You may place the plates in a 25C incubator overnight.

  7. Stack plates with the same type of media and slide the plastic sleeve over the top.

  8. Flip the stack over and seal the plastic sleeve with masking tape.

  9. Label the tape with the type of media, date produced, and name of individual that produced the stack.

  10. Store sealed stacks in the refrigerator until use.  

Chemotaxis Assay

  1. Allow C. elegans to grow on E.coli OP50 bacterial lawn plates for approximately 3 days so they are able to sync life cycles. 

Washing the Worms

  1. Synchronize worms to young adults

  2. Pipette 2 ml of the S Basal onto a 5 cm Chemotaxis plate of staged worms that have just cleared the lawn of OP50 E. coli. Tilt the plate as needed to ensure the worms are washed from the plate surface into the buffer.

  3. Pipette 1 ml of the worm-S Basal solution into a microcentrifuge tube.

  4. Centrifuge for 10 sec using a PicoFuge at 6,600 rpm.

  5. Aspirate the S Basal, leaving the pellet of worms undisturbed.

  6. Add 1 ml S Basal solution to the microcentrifuge tube, invert a few times to wash the worms.

  7. Repeat steps 1.4 to 1.6 another three times.

  8. Centrifuge for 10 sec a fifth time, this time aspirating the supernatant to a final volume of ca. 100 μl.

  9. Pipette 2 μl of the worms onto an NGM plate to ensure there are between 50 and 250 worms in each 2 μl sample. Adjust the concentration of worms in the S Basal as needed by resuspending the worms in a smaller or larger volume of S Basal.

  10. Use the worms in the assay immediately after washing the worms for up to 1 hr.

Plating

  1. Marking the underside of a 5 cm plate, divide the dish into 4 equal quadrants. Chemotaxis agar or NGM may be used. A minimum of 3 trials are required per genetic strain being tested.

  2. Mark a circle of radius 0.5 cm around the origin

  3. Mark a point in each quadrant with either a "T" for "Test" or a "C" for "Control," ensuring that the sites are equidistant from the origin and each other. The points must be at least 2 cm away from the origin. Mark the top left and bottom right quadrants as test quadrants and the top right and bottom left quadrants as controls

Running the Assay

  1. Pipette 2 μl of the worm solution (prepared in 1.8) from the pellet onto the origin (where the two lines intersect).

  2. Immediately after, pipette 2 μl of the test solution (salt solution)  onto the two "T" sites. Likewise, pipette the same amount of the control solution (water) onto the two "C" sites.

  3. Once the worm and odorant drops have been absorbed in the agar, replace the lids and invert the plates.

  4. After 60 min, place the worms in a 4 °C incubator. Only remove a plate from the incubator when it can be counted. Leaving worms at room temperature may allow them to mobilize again.

  5. Record the number of worms in each quadrant that completely crossed the inner circle.

  6. Repeat steps 3.2 to 3.6 using the control solution in both the test and control quadrants. This will serve as the control plate. Three such plates should be made and run to serve as an appropriate control.

Interpreting the Scores using a Chemotaxis Index

  1. Calculate the chemotaxis index using Equation 1. This will yield a chemotactic index between -1.0 and +1.0.

Chemotaxis Index = (# Worms in Both Test Quadrants - # Worms in Both Control Quadrants) / (Total # of Scored Worms)

A +1.0 score indicates maximal attraction towards the target and represents 100% of the Add 2 ml of the polyphenols to the experimental group plates. worms arriving in the quadrants containing the chemical target. An index of -1.0 is evidence of maximal repulsion.

Questions and Answers

 

1. What was the major objective of your project and what was your plan to achieve it? 

After working with C. elegans during the Research elective course as a junior, I found it to be extremely interesting that C. elegans had an intricate neural system and was a model organism for studying human pathology. 

 

2. What were the major tasks you had to perform in order to complete your project?

The major tasks required for completion of my project included the cultivation and synchronization of C. elegans life cycles. This was accomplished by plating the C. elegans on Nematode Growth Medium (NGM) plates along with OP50 E. coli. 

Another major task was effectively and accurately completing the chemotaxis assay. 

 

3. What is new or novel about your project?

My project was novel in the usage of polyphenols to promote neural regeneration in a model organism. The background research that I had performed had shown me that there was very little research done specifically using C. elegans and observing the effects of polyphenols on their behavior. 

 

4. What was the most challenging part of completing your project?

The most challenging part of completing this project was most likely the chemotaxis assay. Specifically, the difficulty of making sure the concentrations of how many C. elegans ended up on each plate was difficult because of the different vendors they were ordered from. However, this was fixed by adjusting the molarity of each S Basal and C. elegans.

 

5. If you were going to do this project again, are there any things you would do differently the next time?

If I were able to do this project again, I would want to use multiple strains of Parkinson’s modeled C. elegans to see if the results varied from strain to strain. I would also like to repeat trials to ensure precision of the results. Another concept I would like to implement into my project is using GFP as a biomarker of neurons and tracking neural growth using a more powerful microscope. 

 

6. Did working on this project give you any ideas for other projects? 

Working on this project really made me think about the field of neural regeneration through food consumption. I would also like to perform an experiment on how different levels of food processing can affect the behavior of C. elegans and the neuron maintenance of the organisms as a model for humans. 

 

7. How did COVID-19 affect the completion of your project?

COVID-19 made the completion of this project a bit difficult, considering I was not able to continuously monitor the growth of the C. elegans. The delivery of some materials was inhibited by both COVID-19 and the inclement weather. Finally, I was unable to contact any professionals on C. elegans and model organisms and have them observe my work and give me feedback due to COVID-19. However, none of my procedure was directly impacted by the pandemic.