Advanced STEM 1 with Scientific Technical Writing is taught by Dr. Crowthers. This was a two-term course that covered everything from research to engineering and scientific technical writing. One of our primary tasks was our independent STEM 1 project; it was a five-month long project that we presented at the STEM fair. On this page, you can explore my project in detail! I also have a project research page(add hyperlink), where you can get a deeper insight into the research stages of my project.

Inducing chronic inflammation in embryonic Danio rerio to assess symptomatic variations in those developing Alzheimer’s.

Abstract

Brain inflammation causes a medley of problems that can progress to diseases such as Alzheimer’s. Through activating microglial cells, proinflammatory cytokines and toxic products are released. For example, head trauma patients experience an increase in amyloid β buildup due to elevated levels of the cytokine IL-1β. The increase of this cytokine induces the expression of IL-6, activating CDK5, a kinase that hyperphosphorylates tau. This cascade leads to the accumulation of paired helical fragments, forming neurofibrillary tangles. The combination of both the amyloid burden and tau tangles causes the death of neuronal cells. Identifying the impact of inflammation on behavior can help diagnose the neurodegenerative disease earlier, resulting in a better quality of life for the patient. The objective of this project is to assess varying concentrations of Pb Oxide, a proinflammatory metal complex, in Danio rerio creating an identifiable behavioral reaction in those who acquire Alzheimer's due to inflammation. To test this hypothesis, zebrafish embryos prior to day 7 were grouped into 4 concentration levels of Pb Oxide, 40 μg/ml - 320 μg/ml. Reflexive behavior declined, asserting the lack of response in stimuli, and operant behavior and locomotor activity also declined, affirming inflammation as a cause of the cognitive decline. The symptoms observed can serve as a baseline for future diagnoses, especially when the patient is known to suffer from chronic inflammation. In the future, behavioral differences between gender can be studied to further specify symptoms. Keywords: brain inflammation, locomotor activity, reflex, cognitive decline

Background

Losing memories with age is a concept that juxtaposes the idea of living through diverse experiences and collecting memorable moments to look back on. However, Alzheimer’s is a neurodegenerative disease that does not give importance to which part of one’s life it is erasing, nor the person it is affecting. In fact, one person every 3 seconds acquires dementia, and there are over 55 million people in the world whose lives are slowly fading due to this disease; statistics show that this number is to almost double every 20 years (Alzheimer’s Disease International, 2021). There are three stages of Alzheimer’s Disease (AD): mild, moderate, and severe, and this disease can only be diagnosed after performing brain scans such as MRIs and CT scans. The disease is typically diagnosed in the mild AD stage when symptoms are not as severe (National Institute on Aging, 2021). In general, symptoms range from memory loss and difficulty completing everyday tasks to seizures and weight loss (National Institute on Aging, 2021). As of current research, all the triggers of Alzheimer’s are not yet established, but it is known that the trigger gives rise to the excess buildup of protein in and around brain cells (National Institute on Aging, 2021). There are two types of proteins involved in the pathology: amyloid β, whose buildup forms plaques in the brain, and tau, whose buildup forms tangles in the brain (NHS, 2021). Eventually, the buildup causes the death of neurons and reaches apoptosis, affecting the memory. The level of each protein in the brain is impacted by environmental factors such as age, family history, and other diseases, where an excess of each leads to Alzheimer’s (National Institute on Aging, 2021). Although there is currently no cure, identifying the symptoms associated with AD early on and starting drug therapy improves the overall quality of life for patients. Once a trigger can be identified, symptoms associated with the trigger can be observed to customize the symptoms for that specific trigger. All the current causes of the disease are unknown, but recently, brain inflammation has been linked to Alzheimer’s. Identifying behavioral changes for inflammation that correlates to Alzheimer’s consequently creates a smaller pool of possible symptoms. The Problem with Symptoms Forms of Dementia such as Alzheimer’s Disease (AD) affect everyone differently, and there is no way to predict how personality and behavior will change. In addition, the mindset of the patient about the diagnosis along with emotional and mental health status can impact the symptoms associated with the neurodegenerative disease (Warren et al., 2021). The discrepancies in the range of symptoms are large, making it difficult to immediately connect to AD. However, there are a few key symptoms of AD that are standard: confusion, memory loss, trouble in decision making, and performing common everyday tasks. Once these common factors are identified, the connection can be made, and brain scans can be done to verify the diagnosis. The main task, however, is identifying one trigger, which is then analyzed to identify the symptoms. The connection As inflammation was identified as a possible cause of Alzheimer’s, a deeper investigation was furthered. There are two types of inflammation: acute and chronic. Acute inflammation is common in everyday life ranging from small injuries to contracting the common flu. White blood cells surround the infected area to speed the healing process, and it is beneficial for the body to fight off the infection as it strengthens the immune system (Harvard Health Publishing, 2020). However, when chronic inflammation, or prolonged inflammation, occurs, the body continues to release white blood cells indefinitely, because the chemicals detect an ongoing attack in the body. Eventually, the immune system attacks the body, because the white blood cells start affecting nearby healthy tissues and organs (Harvard Health Publishing, 2020). Specifically, inflammation in the brain activates microglial cells, which release pro-inflammatory and toxic products such as cytokines (Kinney et al., 2018). For example, patients who suffer from head trauma experience an increase in amyloid β buildup, which occurs due to elevated levels of interleukin 1 (IL-1β), a cytokine that plays a role in regulating immune and inflammatory responses (Dinarello, 1997). The increase of this cytokine can elevate levels of another cytokine IL-6, which then activates CDK5, a kinase that hyperphosphorylates tau (Kinney et al., 2018). The hyperphosphorylated tau then accumulates into paired helical fragments, which go on to form neurofibrillary tangles. The combination of both the amyloid burden and tau tangles causes neuroinflammation to become neurodegeneration and reaches apoptosis or the death of cells. The prolonged-release of cytokines and neurotoxins from microglia activates more microglial cells, and the cycle continues. Since chronic inflammation has recently been identified as a trigger to Alzheimer’s, potential methods of being able to observe behavioral symptoms were researched. Lead Nanoparticles Lead compounds are a known toxic chemical to the human body (Wani et al., 2015). Exposure to these compounds can be fatal or cause irreversible changes. While all the organs are affected, the nervous system is especially targeted. A study conducted showed that exposure to Pb promotes proinflammatory cytokines (Sirivarasai et al., 2013). Specifically, exposure increases the cytokines production of Interleukins 1b and 6 (IL-1b and IL-6), as seen through a study using mice as a model to monitor how exposure to Lead Acetate increased symptoms of a bacterial infection (Dyatlov and Lawrence, 2002). The mice injected with these two cytokines and exposed to Pb showed an increase in symptoms of the bacterial infection caused by Pb, and the results supported that children with high levels of Pb in the blood will exhibit prolonged and worsening symptoms of the bacterial infection. This study showed that Pb exposure does promote proinflammatory cytokines, but another study supports that Pb exposure also affects the expression of cytokine genes (Metryka et al., 2018). Inflammatory responses in the brain tissue of newborns were observed. The blood Pb levels of the mothers were raised to 15–20 μg/dL from the beginning o pregnancy to 21 days after giving birth. When cytokine expression was tested at 21 days after birth, there was a significant increase in the expression of IL-6 levels in the brain. This cytokine, along with IL-1b, are both indicators of a proinflammatory response, supporting that Pb exposure does promote inflammation in the brain, which could consequently lead to Alzheimer’s, as previously mentioned. PbO is a form of Pb that is especially harmful to the human body, affecting the kidney, lungs, liver, spleen, brain, and blood (Dumková et al., 2017). The median blood lead level in individuals from North Carolina with the possibility of exposure to PbO increased from 22 μg/dL in 2012 to 37 μg/dL in 2016, supporting that exposure levels are increasing (Rinsky et al., 2018). Even though exposure levels to PbO is rising, there are not many studies establishing connections of this chemical to inflammation, and, in turn, Alzheimer’s. The relevance of this toxic chemical to the current population and the novelty associated with it were the deciding factors in using PbO for the project.

Procedure

Role of Student vs. Mentor The project took 3 weeks to complete, along with around 3 weeks of practice in the lab beforehand. My mentor helped me set up the Lead (II) Oxide concentrations and the equipment necessary to complete the project. I completed the rest of the project, where I combined the different concentrations of Lead Sulfide with the fishwater solution, took reflexive and locomotor data, measured under the microscope, analyzed the data, fed the zebrafish, incubated them, and properly disposed of them. Equipment and Materials The Acetic Acid, the intracytoplasmic micropipette, the Plugable Digital Viewer Microscope, the culture plate, the incubator, and the micropipette were given by the lab at the Massachusetts Academy of Math and Science. Lead (II) Oxide was acquired by Home Science Tools. The zebrafish embryos were given by the Lawson Lab at the Umass Memorial Lab on a weekly basis. Technique Many components of the procedures in this project were similar to that done by a study (Truong et al., 2010). To make stock concentrations of Lead (II) Oxide, Acetic Acid was mixed with Lead (II) Oxide and was then diluted into the fishwater solutions. The embryos were incubated at 28 degrees Celsius and stored in a 6-well cell culture plate. To insert the concentrations of Lead (II) Oxide, a micropipette with the capacity of 0-10 μg/mL was utilized. An intracytoplasmic micropipette was used to probe the tail of the zebrafish embryos to assess locomotor and reflexive activity, and a Plugable Digital Viewer Microscope was used to take photographs of the embryos. The same grid was placed under the wells every day to maintain constant scale while taking photos. To analyze the images, the software ImageJ was utilized, and the lengths of the zebrafish embryos until 7 days post fertilization was measured to assess malformations. The reflexive data was dichotomous; for the locomotor activity, the distance swum by each embryo was recorded for the varying concentrations. Statistical Tests The Cochran’s Q test was used to analyze the dichotomous reflexive data, because there were more than 3 groups, and there was 1 independent variable, which fits the definition of the statistical test well. Once the Cochran’s Q test was completed, a t-test was used to differentiate between the different concentrations. A one-way ANOVA was done on the distances traveled by the zebrafish embryos because there were multiple groups, and a post-hoc was then completed to differentiate between treated and untreated groups. To analyze the body lengths, the Pearson’s Correlation Coefficient was used to identify whether there was a linear relationship between the body lengths of different concentrations.

Figure 1

Figure 1: Concentration and body length has a Pearson's Correlation Coefficient of -0.9221, which suggests a strong negative correlation. A p-value of 0.025842 is observed, hence the results are deemed statistically significant.

Welcome to STEM1

Inducing chronic inflammation in embryonic Danio rerio to assess symptomatic variations in those developing Alzheimer’s.

Overview

Abstract

Research Proposal and Literature Review

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Background

Procedure

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