Tag Archives: inflammation

Ozone: Protector or Pollutant?

We usually hear that we want more ozone in our atmosphere to protect us from the sun’s ultraviolet rays, but ozone isn’t always a good thing. That protective ozone is found in the stratosphere, while ozone on ground level is a harmful air pollutant caused by emissions from cars and factories. Ozone can do a lot of damage to human lungs, causing shortness of breath, coughing, inflammation and damage to airways, aggravation of lung diseases like asthma, and permanent lung damage. In response to ozone-induced injury, macrophages (immune cells which eat and break down viruses, bacteria, and dead cells) accumulate in the lung and contribute to inflammation and toxicity. Inflammation is important to get rid of any dangerous invaders or cell debris, but macrophages can also have damaging effects in the lungs.

We want to find out what can be done to reduce that inflammation and toxicity, so we are investigating valproic acid. Valproic acid is a fatty acid which has been shown to be anti-inflammatory and an antioxidant. My research project involves testing our hypothesis that valproic acid will reduce lung inflammation and toxicity caused by ozone-induced injury. To evaluate the effects of valproic acid on inflammation and toxicity, I stain thin slices of lung tissue by immunohistochemistry. In immunohistochemistry, the goal is to determine if alveolar macrophages are expressing markers of inflammation or toxicity – the more expression of a certain marker, the darker the macrophage should be stained. We expect that the lungs of mice treated with valproic acid will be less stained than the untreated if inflammation and toxicity are mitigated.

https://www.edf.org/health/why-smog-standards-are-important-our-health

Caption: Smog over LA. Ozone is the main component in smog.(1)

 

Realities of Research

Like the bad and good faces of ozone, doing a research in the lab is slow-going, but also rewarding. The pace is slow because I dedicate a lot of time to troubleshooting the immunohistochemistry process. For each marker of interest, the protocol needs to be optimized. This is time-consuming because it means going through the immunostaining process repeatedly, changing small details each time. It was especially frustrating when the results were not what we expected. When our controls were repeatedly turning out different from how they had looked in previous experiments, we had to figure out if it was the fault of the sample, a detail in the protocol, or the antibody. I’m currently still working on figuring out the discrepancy by testing samples from other labs and different antibodies. If it’s the samples that are faulty, we will put a hold on the immunohistochemistry until we can use the samples from an animal exposure we have planned in a couple of weeks. If the antibodies are the problem, we will order new ones. If I’m doing something in the protocol incorrectly, my research mentor will watch me go through the steps and find out. This complication has been slowing down our progress, but it’ll be rewarding to finally figure it out and get data.

Life of a Scientist

Even excluding the satisfaction of getting data, I feel like I’ve grown a huge amount working as a scientist this summer since it was completely new for me. It’s my first experience working full-time, in addition to taking place in the unique environment of a research lab. I was happily surprised by the amount of flexibility in schedule – each person comes in and leaves when they need to, depending on the work they need to get done that day. Some days are a typical 9 to 5, some might be much shorter, and some might go late into the night. It can become overwhelming meeting new people, catching up on literature, and learning new lab techniques. However, it’s also satisfying to soak up so much new information so quickly and see myself developing as a scientist and a student every week. In my experience so far, the best part of working full-time is the people I have been able to get to know. Seeing the lab tech, the faculty, the grad students, the undergrads, and the high school students every day gives me the chance to really learn about what they do inside and outside the lab. Because of them, coming into the lab every day is welcoming and exciting, which makes all the difference when I’m frustrated with my experiments. Working with them is easy and most of all, fun, and I’m grateful I was able to do research with such encouraging and friendly people.

 

References:

  1. Why smog standards are important for our health. (2018). Retrieved July 27,2018, from https://www.edf.org/health/why-smog-standards-are-important-our-health
Jordan Lee is a junior studying molecular biology and biochemistry at Rutgers University in New Brunswick, NJ. She is a 2018 Undergraduate Summer Research Fellow, funded by the APS. Jordan is working in Dr. Debra L. Laskin’s lab at the Ernest Mario School of Pharmacy at Rutgers. After graduation, she plans to continue doing research and exploring her interests in healthcare and science.
My Experience Interpreting Oxidative Stress and Inflammation in Hypoxia Using Lipid Metabolism

This summer I worked on a metabolomics project surrounding the effects that hypoxia, or deficient oxygenation, has on oxidative stress and inflammation. I used metabolomics, or the study of the functional molecules in the body, to interpret the molecular changes that eventually lead to physiological complications. Currently, we know that oxylipins, biological molecules that are metabolized from polyunsaturated fatty acids (PUFAs), are markers of oxidative stress and inflammation in conditions such as hypoxia (1). My lab extracted venous plasma from fetal and newborn sheep that were living at high-altitudes, as hypoxic conditions can be simulated by high-altitude living. By running tests that quantified the oxylipins in fetuses and newborns, I was able to distinguish which metabolites were prominently affected by hypoxic conditions. Later, I was able to find pathways, tracing how certain PUFAs were metabolized and from which PUFAs certain oxylipins were derived. Based on these relationships, I aimed to find possible roots for the oxidative stress and inflammation caused by chronic hypoxia.

These findings highlight our understanding of lipid metabolism as it is affected by high-altitude hypoxia. This study has the potential to help us develop treatments that target inflammatory pathways induced by pre and post-natal hypoxia. For instance, the enzymatic pathway CYP, which metabolizes PUFAs proved to play a large role in the production of oxylipins that were affected by hypoxia. Targeting this pathway early in the womb may help prevent lung dysfunction that may develop just after birth.

An area in my research that I found difficult was the dense literature. At first sight, it was intimidating‒ scientific jargon and compound nomenclature most of all. I realized that as I started connecting terms to function‒ associating oxylipins with potential roles in the body was now feasible. The truth is, it takes time and understanding to grasp the material, but the more I read and the more I searched, the less intimidating it all felt. Around the lab, there are several skills to master‒ several of which consist of success and failure. For instance, I had a hard time developing networks for my metabolites and working with statistical software early on in my research; this is now something I wish to improve. Often, I received results from my data that I did not expect and it reminded me how difficult it was to remain impartial. I ran into a list of questions that, over time, clarified and narrowed what in fact my research would delineate.

I could summarize the life of a scientist in one word: unpredictable. It surprised me how difficult consistency actually was with data. That being said, it is a huge task to filter data and focus on only a few aspects of it; everything seemed important. Moreover, a reliable, cooperative lab team is a vital component to a scientist’s life in the lab. While not everyone is specialized in the same subject or project, a team creates a supportive environment where we feed off of one another’s knowledge and work in collaboration for the interest of science.

References

  1. Gabbs M, Leng S, Devassy JG, Monirujjaman M, Aukema HM. Advances in Our Understanding of Oxylipins Derived from Dietary PUFAs. Adv Nutr 6: 513–540, 2015.
Vanessa Lopez is a junior Biochemistry major at Occidental College in Los Angeles, CA. She is a 2017 Short-Term Research Education Program to Increase Diversity in Health-Related Research (STRIDE) Fellow. She works with Dr. Sean Wilson in his lab at Loma Linda University in Loma Linda, CA. Vanessa’s fellowship is funded by the APS, as well as a grant from the National Heart, Lung and Blood Institute (Grant #1 R25 HL115473-01). Her research is also supported by NIH grants HD083132 [LZ], 1U24DK097154 [OF] through Dr. Wilson’s lab. She is interested in endocrinology and dietetics. Her plan is to go to medical school after graduation.
Learning from Obstacles in Science

The Western diet is high in fats and sugar and can lead to an increase in metabolic diseases, which cause a chronic state of peripheral inflammation (1). My project this summer aims to observe the effect of diet on brain inflammation. We used a mouse model of tagged peripheral monocytes (3). Monocytes turn into macrophages, which target inflammation in the body and brain (2). These mice were fed either a diet high in fat and fructose or a normal diet for 5 weeks. Then the blood from their brain was washed out, and the brain was sliced. The slices were stained for the genetic tag for the peripheral macrophages. Peripheral macrophages found in the brain suggest that chronic inflammation weakens the blood-brain barrier, allowing peripheral macrophages to cross where they increase brain inflammation. This may cause damage and may have links to diseases such as Alzheimer’s Disease and Parkinson’s Disease, which show increased inflammation in the brain (4). This project would further support the idea that a healthy diet could be a key factor in prevention of brain diseases.

This project, as well as science in general, had many obstacles that I had to overcome. Originally, I planned to analyze a different genetic mouse that modeled Alzheimer’s disease. Those mice were also going to be fed a high fat high fructose or control diet, and were going to be compared to see if there was an increase in peripheral macrophages in the brain in diet treated mice. However, those brains didn’t have the blood cleared from the brain, which limited our ability to see the stain. To overcome that problem, we used the new mouse type that had peripheral monocytes tagged, which had the blood removed from it. With this new mouse model, I would have a smaller number of animals, but I could better test my hypothesis.

I enjoy the day-to-day life in research. I was expecting it to be somewhat repetitive, but that was far from the case. I had many problems that I had to solve and was constantly learning, which made the time fly by. My day was broken up by working on different parts of my experiment, writing and reading literature, and meeting and talking with my lab members. The best part for me was that I constantly learned new things. There were many hiccups in my summer experience, which were disheartening at times. However, solving these problems and further learning more made it rewarding as well. The Tansey lab has many members who have been very helpful in solving these problems. I enjoyed being a part of a larger team, as there were so many projects going on that I could learn from.

References

  1. De Sousa Rodrigues, M. E., Bekhbat, M., Houser, M., Chang, J., Walker, D., Jones, D. P., Oller do Nascimento,C., Barnum, C. J. & Tansey, M. Chronic psychological stress and high-fat high-fructose diet disrupt metabolic and inflammatory gene networks in the brain, liver, and gut and promote behavioral deficits in mice. Brain, Behavior, and Immunity 59: 158-172, 2017.
  2. Khoury, J. E., Toft, M., Hickman, S.E., Means, T. K., Terada, K., Geula, G., & Luster, A. D. Ccr2 deficiency impairs microglial accumulation and accelerates progression of Alzheimer-like disease. Nature Medicine 13: 432-438, 2007.
  3. Saederup, N., Cardona, A. E., Croft, K., Mizutani, M., Cotleur, A. C., Tsou, C.-L., Ransohoff, R. M., & Charo, I. F. Selective chemokine receptor usage by central nervous system myeloid cells in CCR2-red fluorescent protein knock-in mice. PLoS ONE 5: e13693, 2010.
  4. Selkoe, D. J. The therapeutics of Alzheimer’s disease: where we stand and where we are heading. Annals of Neurology 74: 328-336, 2013.
Lindsey Sniffen is a senior majoring in Neuroscience and Behavioral Biology at Emory University in Atlanta, GA. She is a 2017 Integrative Organismal Systems Physiology (IOSP) Fellow in Dr. Malu Tansey in the Department of Physiology at Emory University in Atlanta, GA. Her fellowship is funded by the APS and a grant from the National Science Foundation Integrative Organismal Systems (IOS) (Grant #IOS-1238831). After graduation, she plans to pursue a Ph.D. in pharmacology, and then work in the pharmaceutical industry.
Impact of Injury on Inflammation

For my research project, I will compare the levels of a known marker of inflammation in and around motor neurons in rats with and without cervical spinal cord injury. We will examine rats with chronic C2 incomplete spinal cord injuries, and compare them to uninjured tissues. We will be examining the frozen and preserved tissue under a microscope to quantify the different levels and locations of inflammatory markers at the different time points. The results of this experiment are important because they will enable us to better treat those who have suffered from the devastating effects of spinal cord injuries. This experiment is necessary to determine if p38 MAP kinase (a specific known marker of inflammation) is activated following spinal cord injury. The results will allow us to determine how to proceed in our search for successful rehabilitative treatments for patients. If p38 MAP kinase is activated following injury, it may call for future studies to investigate treatment of this specific cause of neuroinflammation in order to improve the outcomes of the rehabilitative treatments our lab is studying. Neuroinflammation can decrease the positive effects of rehabilitative efforts, and therefore is something we need to study so we can reduce this inflammation and better treat those who are suffering from spinal cord injury.

Life in the Lab

My experience in the lab was very educational. I did not realize how many different things actually go into research. I became familiar with behind-the-scenes tasks that you do not realize need to be done. For example, we spent a large majority of our time sectioning tissue, which I never realized would be such a large and time-consuming part of the experimental process. In addition, I became independent in many different techniques and procedures used in our lab. I learned proper animal handling and care, the methods used for immunohistochemistry, as well as proper imaging strategies and techniques on the Keyence microscope that we use in our lab. I also learned how to problem solve when problems arose. I found that one of the biggest challenges in research is how time-consuming and detail-oriented everything is. It is necessary to plan very far in advance and plan other aspects of your day around what is needed in lab. I was studying for the MCAT while completing my research project this summer, and I found it very difficult to dedicate time to studying. However, I quickly learned to manage my time wisely and study during down-time in lab and in the evenings. I believe this skill is not only valuable for research, but will also help me throughout my life.

Although what I did day-to-day varied, there were certain tasks that remained constant. For example, animal care and running exposures was something that needed to be completed by someone every day, so I was usually around to help out with those two things. In addition, the weeks were organized in a way that there usually was not more than one big task going on at a time. For example, there were weeks focused on surgeries, as well as others focused on perfusions and harvesting. What I did between rounds of animal care throughout the day varied depending on the week and what needed to be completed. Some days were filled with staining, while others dealt with microscopy. In my opinion, the best part of working in a lab was how often you were able to see your hard work pay off. Although the experiments tend to take at least a few months to complete, there are many milestones where you begin to see the outcomes of all of your hard work. Personally, I thought the hardest part of my time in research was not having set-in-stone days. Your schedule can vary every day depending on the point of the project you are in and what needs to be done, so you need to be able to adjust your plans and schedule around your lab responsibilities.

One of the best parts about working in a lab is being a part of a large team with a common goal. It is much more rewarding to accomplish a goal when everyone is working on it together in my opinion, and it’s nice to always have people around who are willing to help you and your project succeed. Research is usually not a one-person job, and for good reason; there is so much that goes on to ensure that a project is successful, and everyone in lab is needed. Throughout my research experience, I have developed a deep appreciation for how important research is to the functionality of many different aspects of society. Advancements in everything from technology to medicine would not be possible if scientists were not working hard in lab each and every day, and I am glad I will be able to take this appreciation for research into my future career. Research is a tough task, but it is truly life-changing in more ways than one.

Ashley Holland is an undergraduate at the University of Florida in Gainesville, FL. She is working in the lab of Drs. Gordon S. Mitchell and Elisa Gonzalez-Rothi at the University of Florida under the UGREF fellowship funded by APS. After graduation, Ashley hopes to attend medical school and use her skills acquired during her research experience to further the medical field and help her patients in new and innovative ways.