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Antioxidants can be used to help prevent neurodegeneration and reduce ROS species associated with Post traumatic Stress Disorder (PTSD) in a C. elegans model. 1/11 people are diagnosed with PTSD and this number is increasing. PTSD affects day to day life, since flashbacks and nightmares may occur at any time, causing a flight or fight reaction. Previous studies have shown a link between oxidative stress and PTSD, as higher levels of oxidative stress in the brain correlate with more neuroinflammation. It was predicted that if the natural substances work then the reactive oxygen species (ROS) of the test group and their avoidance behavior caused by a trigger molecule should be similar to non-PTSD worms. The N2 C. elegans strain was put through an avoidance assay to simulate PTSD by having a trigger molecule: diacetyl. There were 3 paradigms, 2-32˚ C degree water baths (1 as temp control) or 20˚ C. Diacetyl is an attractant for the worms, but the assay conditions the 32˚C degree group to avoid it. Avoidance behavior is classified as one movement backwards. The antioxidant mixture was mixed with LB and E.coli, and then plated as a food source on NGM. ROS was measured using the LD1171 strain put through the same conditioning, a fluorescent microscope, and Image J software. The 32˚ C degree and diacetyl group that had antioxidants in their food source avoided a statistically smaller average amount of diacetyl drops compared to either control group without diacetyl present in the conditioning. The conditioning did not cause the worms treated to make an association between the warm water environment and the diacetyl, which means the treatment worked, preventing avoidance behavior and PTSD symptoms. This study lays the groundwork for investigating the relationship between associations and PTSD, and future actions include investigating epigenetics to see if PTSD behaviors and ROS expression will reoccur in the offspring.

Post-traumatic Stress Disorder (PTSD) is a psychiatric disorder that occurs in people who have witnessed a traumatic or life-altering event. Some people may be more susceptible to having PTSD because of a recently discovered genetic mutation (Louge et al., 2012). PTSD affects day to day life, since flashbacks and nightmares may occur at any time causing the person to relive the initial trauma. The most common symptoms and behaviors associated with PTSD are fight or flight behaviors (avoidance), mood swings, agitation, anxiety, and low energy (American Psychiatry Association, 2020).About 1 out of every 11 people will be diagnosed with PTSD. Current treatments include cognitive behavioral therapy, along with medications. These medications are often antidepressants, and specifically SSRIs (selective serotonin reuptake inhibitors) are most often prescribed (American Psychiatry Association, 2020). However, cognitive therapy can take years to help patients manage their PTSD symptoms and SSRIs can cause certain liver metabolism functions to not occur at all and these antidepressants can easily build up in a human system causing withdrawal effects (Lane et al., 1995). Additionally, neither antidepressants or therapy targets and reduces the amount of radical oxygen species and oxidative stress present during one’s brain in a PTSD episode or anytime.

Recently, evidence is pointing to the possible involvement of oxidative stress in PTSD. Oxidative stress is an imbalance between free radicals and antioxidants in your body; free radicals are oxygen-containing molecules with an uneven number of electrons. The uneven number allows them to easily react with other molecules and cause unwanted or unnecessary chemical reactions (Miller et al., 2018). In a study, Logue et al. found a genome-wide significant association between a single nucleotide polymorphism (SNP), the most common genetic mutation where one nucleotide is deleted, in the Retinoic Acid Orphan Receptor Alpha gene (RORα) and a diagnosis of PTSD among veterans (Louge et al., 2012). It is activated during oxidative stress and serves to protect neurons by increasing the expression of other genes involved in the clearance of reactive oxygen species (ROS). ROS and oxidative stress have been known to trigger the Sympathetic Nervous System, responsible for causing "fight or fight" behaviors associated with PTSD (Schiavone et al., 2013).

Previous studies have shown the effectiveness in antioxidants at reducing oxidative stress. Ahmed et al., 2019 continues, finding that antioxidants are able to balance ROS molecules by donating electrons, to help stabilize the oxygen species. When the molecules become stable, they no longer pose a threat to the nervous system. For this project, blueberry extract’s most abundant antioxidant malvidin, and pomegranate’s most concentrated antioxidant punicalagins were chosen. Only two antioxidants were chosen because of how strong they are, as well as this study can relate the 2 specific antioxidant molecules and their concentrations to the results, instead of broadly discussing blueberry and pomegranate’s effects.

PTSD Avoidance Assay
The avoidance assay was used for our treated worms and non-antioxidant treated worms. After an hour of conditioning was complete, the plates were transported to an assay room with controlled temperature and humidity. To test the avoidance, a repellent (which was an attractant initially) was used to see if the conditioned worms with diacetyl would avoid the once attractive diacetyl, as well as the reactions to diacetyl. Then, a capillary tube was attached to a mouth pipette, and used to pick up either the solvent control or diacetyl. A small drop of either solvent control or repellant was placed at the head of the worm, and at least 15 worms per plate and per liquid of solvent control or diacetyl were tested. If the worm propagated backwards before 5 seconds passed, it was considered avoidance. The percent avoidance for the diacetyl and solvent control was recorded for each group.

ROS Analysis Technique
In order to analyze the ROS present before and after the conditioning depending on treatment, the LD1171 C. elegans strain was utilized because of the GFP it expresses in the location of ROS molecules. The conditioning method listed above was utilized, but instead of testing avoidance, the worms were immediately moved to the imaging room where a fluorescent microscope was used to observe fluorescence and take pictures of about 15 worms per conditioning trial.These images were then uploaded to Image J and converted to grayscale, and the ROS of each individual worm from the pictures were analyzed for average gray value. This index represented the concentration of the fluorescent (or lighter gray) in the body of each of the worms. After, the background of each of the worms was also analyzed, and given a value. Since the background was darker gray because it did not have GFP, the index was lower. These two values were then subtracted, worm index - background index, and the average of these differences were used to compare treated vs non treated groups.

Focusing mainly on Figures 1 and 2, we can see that the PTSD only group was very significantly different than all the other groups in both graphs. In Figure 1, looking at the PTSD and antioxidant treated group, there was less than a 3% percent difference in avoidance compared to the control and antioxidant control, yielding a not significant difference as indicated by the p-value of 0.075 and 0.15. This demonstrates that the experimental group treated with both antioxidants and the PTSD conditioning displayed similar avoidance behaviors as the control groups. Additionally, the difference between the control and antioxidant avoidance indexes was not found significant.

In Figure 2, the PTSD plus antioxidant treated group had a significant difference in the GFP expressed compared to the antioxidant control, indicated by the p-value of 0.021. This demonstrates that the experimental group treated with both antioxidants and the PTSD conditioning displayed statistically different GFP expression than 1 of the control groups. However, the difference between the PTSD plus antioxidants, control, and antioxidant control was not found to be significant by an ANOVA test that resulted in a p-value of 0.183. Furthermore, the difference between the antioxidant plus PTSD group and control group was not found to be significant. Additionally, the difference between the control and antioxidant avoidance indexes was not found significant.

Future research includes sequencing any changes in the C.elegans genome or any neural death that may occur because of the stress and conditioning of the PTSD simulation. Additionally, looking into the generational effect the conditioning may have on the offspring and future generations of the heat-shocked worms. An extension of this project is looking at different heat-shock assays to simulate PTSD, such as a chemotaxis assay. Lastly, gaining more data would be ideal just to gather more evidence between the relationship and associations between the conditioning, avoidance, and ROS.

This study shows that antioxidants can be used preventatively, as a food supplement, to help prevent or limit the impact that PTSD symptoms may have. The main behavioral symptom being avoidance behavior, with a secondary symptom of reactive oxygen species. In the study, it was shown that ROS was decreased and GFP expression decreased in the treated group. Additionally, overall avoidance behavior caused by C.elegans’ sympathetic nervous system became similar and not statistically different from the avoidance behavior of the control groups. This research has additional implications on the world today, because of the epigenetic opportunity it holds. If it can be proven through a generational study using C.elegans and the conditioning of future offspring also avoid diacetyl, without conditioning, then studies that hypothesize a genetic linkage between mental illness could be validated and then other animal models could be used as well. As the conversation about mental health becomes more normalized, and the push for natural and holistic treatments becomes more popular, antioxidants hold the key to treating oxidative stress.