Blog Post 2: Nearing the End of My Research Journey

Hello everyone! I hope you are all doing well. As I am nearing the end of my research, I wanted to sahre a few updates with you all.

First of all, let me reiterate what my research project was about.

Many articles online claim that Green tea is one of the best brain healing foods after a concussion (Flint Rehab, 2020), however I couldn't actually find a study or a journal article looking at one of the most prominent amino acids in green tea specifically, called L-theanine in the Traumatic Brain Injury context. There is research about another component such as Epigallocatechin-3-gallate, but L-theanine is also an essential component that is worth looking at. It has wonderful benefits in other injuries such as cerebral ischemia-reperfusion (a stroke based brain injury), and even during Traumatic Spinal Injury in rats. Last year I did a project with my biotechnology group and we found that the group of C. elegans with L-theanine administration experienced worse effects as shown through their axons in the brain. This year, I wanted to take a step back and specifically focus on the RECOVERY in C. elegans due to L-theanine, and attempt to explore this amino acid once again.

In a broad sense, my methodology includes raising the age synchronizing the C. elegans, to keep them at a constant age and to let the worms grow for 72 hours. For their last 24 hours, I allocated the worms to their specific groups as shown in the image below. The respective groups were administered TBI and L-theanine 24 hours before visualization and data collection. At the beginning of my project, I was also planning on imaging an L-theanine control group however due to my setbacks (will be discussed later), and limited time frame, I had to cut down the L-theanine control group as it wasn't a gigantic focus on my project (Figure 1).

Figure 1: Here is a visual of my different groups during this experiment. I will be comaring these groups together and visualizing 10 worms from each group (a total of 30 data points).

For the drug administration, I poured my own plates with the required NGM-LITE agar, and mixed in the L-theanine concentration of 100 ug/ml into the agar. Then I used the dead plate method of drug administration, since it was the most effective (Zheng et al., 2013). Below is how the E. Coli culture (food source) was made utilizing this specific method (Figure 2).

Figure 2: Here are the methods of making E. coli culture utilizing the dead plate methods. This was essential for the L-theanine absorption into the C. elegans.

For the actual portion of TBI Induction (Figure 3) and Visualization (Figure 4), methods are placed below (Cooduvalli et al, 2021)

Figure 3: These are the methods for Traumatic Brain Injury in C. elegans developed by a previous Rock Canyon Biotechnology student, Zoe Zizzo. Her methods are used in the John Hopkins Lab (Cooduvalli et al., 2021).

Figure 4: These are the visualization methods developed by Acelyn McGowan, Surbhi Cooduvalli, and Joanne Paraiso with using the EVOS microscope in the RCHS Biotechnology lab (Cooduvalli et al)

A slight change that I made to the methods of TBI above is instead of just pipetting the 10 ul of C. elegans into another tube with two 1mm glass beads, I pipetted them 10 ul of C elegans into 5 centrifuge tubes with glass beads each. This was just to make sure I got as many worms as possible and allowed them to experience TBI. This was because the first time I conducted TBI I was in a rush and my resulting plate had no worms collected from TBI that I could visualize. Therefore, I became much more careful and I took more caution when conducting TBI. This is just a broad overview of my methods, but I will be posting my paper of specific methods later on this year for you all to reference!

Now, let me talk about some roadblocks.

1. The major one was Time. I ran into some problems with my age synchronization and TBI induction of the C. elegans. Since I had a specific time frame of letting the C. elegans grow for 72 hours, and administering them with L-theanine and/or TBI for 24 hours, I could only visualize one group per week. Since I had other commitments outside of school such as TSA I also struggled to get my research finished in time. Furthermore, the visualization process takes almost an hour for visualizing 10 worms, so within the time frame I was given, my data group was limited.

   1. The age synchronization originally did not work since there were no worms left on the plates after age synchronization. This process involves dissolving the worms in a bleach solution so only the eggs remain and hatch to keep the ages constant. I hypothesize that this was because I did not wait long enough for the bleach solution to dry off before sealing the plate, or that the drop I placed was too big. In future trials, I made sure to be extra careful about the time and the size of the drop.

   2. With TBI induction, I usually lose some of my worms through the pipetting process, but I counteracted this by instead of just conducting TBI with one tube, I used five.

With my progress in Data Collection: I finished!! Here are some pictures below! (Figure 5) I will not disclose what groups these images are from right now since I still need to conduct my double blind analysis.

Figure 5: Here are some of the images of the C. elegans collected through the GFP EVOS microscope using the visualization methods from above. The lines within the C. elegans are called axons (part of a nueron), and they often break during TBI. In last one, you can see multiple breaks in the network. The middle one has minimal breaks, but still includes two bright swellings in the axons.

I do need to begin analyzing my data and hopefully I will have it done by the end of spring break. I have two analysis methods, including a double blind analysis and a pixel count. For the double blind analysis I will take two people and give them a shuffled slide deck of the images and ask them to rank the images of worms using the scale provided below.

Figure 6: This is the visual representation of the neuronal damage scale in which the data will be qualitatively assessed. It was developed in Zoe Zizzo's TBI Project (Zizzo et al, 2019).

The other analysis will be an Image J Pixel count that will count the number of green pixels in an image with the intensity greater than 54. Typically healthier worms without TBI have a higher pixel count than worms with TBI.

As soon as I complete analyzing the data, the results will be ready and released.

Overall, the research is aimed at identifying if L-theanine can provide a more cost effective recovery option for those who undergo Traumatic Brain Injury. Traumatic brain Injury often occurs in developing countries and is an expensive endeavor, but natural alternatives found in green tea an be highly beneficial. As this amino acid is gaining traction in the scientific world, I hope my results and research can add on further discussion of L-theanine in TBI. However, it is still important to remember that my research is done on a small model organisms and connecting the results to humans are only a possibility but outside the scope of my research. Future research needs to be done to explore exactly howthis drug works during brain injury. Furthermore, due to my limitations in time and small sample size, the results of this project can be taken with caution.

For future teams that want to continue research in C. elegans, I wish you all the best of luck! In this project, I did find that the dead plate administration method often yielded better results in imaging through the EVOS microscope if you wanted to take a note of that!

Another update that I wanted to share with you is that during this year I shared my three minute presentation with the class. Here is the link to the presentation: https://docs.google.com/presentation/d/1i2EZbmRBN4uuJLtDPh3FiGDBEArpym6y7ElBNMLy1yQ/edit?usp=sharing

During this presentation I got practice presenting in front of my peers and I received feedback on how to present for the final presentation. I will post the final presentation soon!

Stay tuned for future posts about results and how that ties back to our world! I will also be posting my methods soon for any future researchers!

Thank you so much for all of your support!