Monthly Archives: December 2017

Post Spinal Cord Injury Female Mice have a Higher Concentration of Glial Cells

In the laboratory this summer, I studied one of the myriads of cell responses that occur in the spinal cord post-injury. The cells that I studied were microglia, which play a role in cleaning up debris and acting as an immune response. It has been found that post-SCI (spinal cord injury) female mice have better functional recovery than their male counterparts. Scientists studying SCI’s are currently researching possible causes for this difference in healing. My project focused on whether male or female mice have a higher microglia response post injury. The tissue I analyzed was collected from male and female mice 42 days post-moderate SCI. I first had to stain the spinal cord sections with eriochrome cyanine, which shows the degree of degeneration the spinal cord underwent post injury. Immunohistochemistry was then performed on the tissue, which is a form of staining that binds fluorescent antibodies to the cells you are looking for. Because of the fluorescence that is attached to the cell, you can then use light to illuminate the microglia and then image the cells. To get the most accurate comparison, I selected a portion of the spinal cord that I could identify in every animal and quantified the cells in just that area. My data concluded that female mice have significantly (p: .0187) less microglia.

Realities of Research

This was my first time taking part in research in a professional lab setting and there were a few things that surprised me, but for the most part I knew relatively what I was getting into. I think the most surprising thing was that there was a lot of sitting around and waiting for either results or for my slides to dry. As a student that works better when pressurized, I feel that this aspect will be the biggest barrier keeping me from going into a research career. There were also a lot of issues that I had with the tissue itself and aspects that hindered my ability to analyze my results.

My responsibilities changed from day to day, whether I was sitting at a desk and reading for hours straight or doing a stain. I also spent a lot of time learning about lab equipment and how to safely complete an experiment. I think the best part was when I was able to work by myself and take ownership of my own project. However, there were many times that I was lost or confused and really needed help. The worst part of this research was all of the down time that I had. Working as a lab team was very interesting because there were always people around that had something interesting to contribute to my project. However, there were also issues with finding the specific person that could help you with a part of your project.

Julie Wilson is a senior majoring in Biology and Chemistry at Baldwin Wallace University in Berea, OH. She is a 2017 Integrative Organismal Systems Physiology (IOSP) Fellow working with Dr. John C. Gensel at the University of Kentucky in Lexington, KY. Julie’s fellowship is funded by the APS and a grant from the National Science Foundation Integrative Organismal Systems (IOS) (Grant #IOS-1238831). After leaving Baldwin Wallace University, Julie plans to attend medical school and pursue a career in pediatrics working with queer and diverse youth.
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.
Science That Gets Your Blood Racing

Our lab studies cardiovascular diseases such as high blood pressure (hypertension), which affects roughly one third of American adults and puts them at a higher risk for heart disease and stroke, both of which are leading causes of death in the United States [1]. One of our overarching questions addresses the role that the tissue surrounding our blood vessels, or “perivascular adipose tissue” (PVAT), plays in obesity-related hypertension. PVAT contains various types of cells, including adipocytes (fat cells), endothelial cells, macrophages, lymphocytes, fibroblasts, and more [2]. We have previously shown that PVAT contains functional norepinephrine, a signaling molecule that constricts blood vessels and thus increases blood pressure [3]. My project addresses where and how PVAT actually stores this norepinephrine using the PVAT surrounding normal rat mesenteric resistance vessels- the small arteries and veins that branch into the small intestine and are important for blood pressure regulation. We hypothesize that it is specifically the adipocytes in PVAT that store NE, and that they use the vesicular monoamine transporter (VMAT) to do so. A better understanding of this mechanism is important for the future development of treatments for obesity-related hypertension.

[4] Sprague Dawley Rat. Photo Credit: Charles River

Realities of Research

When I first joined the Watts Lab a little over three years ago, I was a wide-eyed freshman who had never even used a micropipette before. Since then, my wonderful mentors have trained me in methods such as immunohistochemistry, brightfield and fluorescent microscopy, cell culture, handling and euthanizing rats, performing dissections, and isolating adipocytes from rat PVAT. More importantly, through these lab experiences, I have learned a scientific way of thinking and hypothesizing. This has been essential for my research project because it was incredibly challenging to develop a new protocol for the functional experiments using live adipocytes. It took over a year and endless troubleshooting for me to get to a point where I had a working procedure, and even longer to replicate the experiments to obtain a large enough sample size. One major obstacle was that freshly isolated adipocytes do not attach well to surfaces such as a microscope chamber slide. Additionally, one of the drugs I tested is only soluble in ethanol, which was toxic to the cells at most concentrations, so we had to find an alternative compound to use. Through trial and error, we have designed methods to effectively isolate, treat, and image PVAT adipocytes for this application. I am also very proud of the protocol we developed to quantify the fluorescent intensities of the cells I imaged, as it is crucial to analyze and present data in as objective and consistent a way possible. I was able to present our work in San Francisco at Council on Hypertension this past September, which was truly an incredible experience.

Conducting research in a lab has been one of the most rewarding adventures of my life. There is a certain thrill in knowing I am working to answer a question that no one else in the world is investigating in the same way. There are definitely setbacks that can be difficult to deal with, such as antibodies that do not work, cells that die for seemingly no reason, or not knowing how to begin designing an experiment you have in mind. However, this is why I love being a part of the scientific community- I have the opportunity to collaborate with the other scientists in our lab and department, or even at other institutions, to gain insight into how to better approach a research question. In addition to the hard work any researcher has to put into his or her own project, I believe it is this enthusiasm to help one another that ultimately allows all of us to be successful. Oh, and a little bit of luck never hurt anyone!

References

  1. High Blood Pressure Fact Sheet [Online]. Centers for Disease Control and Prevention. Centers for Disease Control and Prevention: 2016. https://www.cdc.gov/dhdsp/data_statistics/fact_sheets/fs_bloodpressure.htm [14 Jul. 2017]. 
  2. Miao CY and Li ZY. The role of perivascular adipose tissue in vascular smooth muscle cell growth. British Journal of Pharmacology 165(3): 643-658, 2012. 
  3. Ayala‐Lopez N, Martini M, Jackson WF, Darios E, Burnett R, Seitz B, Fink GD, Watts SW. Perivascular adipose tissue contains functional catecholamines [Online]. Pharmacology Research & Perspectives: 2014. http://onlinelibrary.wiley.com/doi/10.1002/prp2.41/full [14 Jul. 2017]. 
  4. CD® IGS Rat Crl:CD(SD) [Online]. CD® IGS Rat | Charles River. http://www.criver.com/products-services/basic-research/find-a-model/cd-igs-rat?loc=US [14 Jul. 2017].
Maleeha Ahmad is a senior majoring in Genomics and Molecular Genetics at Michigan State University in East Lansing, MI. Her Summer 2017 Undergraduate Research Excellence Fellowship (UGREF) gave her the opportunity to continue working in Dr. Stephanie Watts’s lab at Michigan State University, where she has been conducting research for the past three years. Maleeha’s fellowship is funded by the APS. After graduation, she plans to attend medical school and be involved in clinical research.
Amniotic Membrane Supplementation in Rotator Cuff Reconstruction

Amniotic membranes have been of recent inquiry in the surgical world because of their composition of stem cells. These stem cells can differentiate into the desired type of cells in the body. In this study, amniotic membranes were placed on the insertion of the rotator cuff muscles after rotator cuff surgery to see how this affected recovery time. In order to determine this, the strength of the individual muscle fibers was determined using the single fiber isometric strength method. This method involves pulling out a single muscle fiber from a bundle of fibers obtained from the rat, tying it onto the apparatus and exposing it to high levels of calcium in order to allow for a contraction to occur. After determining the cross-sectional area and the peak isometric force, the specific force (the force per cross-sectional area) can be calculated in order to compare the relative strength of fibers from different samples. This study consisted of four different groups; uninjured control (no surgery performed), control repair (no amniotic membrane supplementation), injury only (no rotator cuff surgery performed), and experimental repair (surgery with amniotic membrane supplementation). There were six fibers obtained from each rat sample with 8 rats per group totaling 192 fibers. Once the specific force is determined for every group, comparisons were made to see if the amniotic membrane supplementation helped restore the specific force of the rats 4 weeks post intervention. This is applicable to human rotator cuff surgeries because it could help patients recovering from this surgery and decrease the recovery time. This would allow for these individuals to return to their normal activities more quickly. In addition, this method can be used in multiple different surgery sites to help improve recovery time.

Realities of Research

This project got frustrating at times because of the variable conditions of the fibers. There were some bundles that contained large, healthy fibers and some that contained extremely small and frail fibers (size comparison can be seen in images 1 and 2). While we did not know what groups these fibers were a part of because of the double-blind format, it was possible to infer which groups these fibers were from. Another interesting thing about this study is that it took three weeks to train for this technique. It started with learning how to tie the minuscule ties used to tie the fibers and then learning how to tie them on a practice machine. We practiced on separate machines because of the high probability we would break the real apparatus if we started on that. However, once data collection started, around 12-18 fibers were run each day and real progress was made. It was also interesting to see the variability of specific forces in a given bundle. This is something that we were not expecting, but was most definitely present. Luckily, the research question never changed, but there were many times that we would have to re-run fibers due slippage, ripping or any other issues that presented themselves.

The difference in cross sectional area for given fibers from different rats.

 

 

 

 

 

 

 

 

 

Day-to-Day Life of a Scientist

Life as a researcher was much more relaxed than I initially thought it would be. The arrival and departure time for each day was variable. While it was expected that you completed all of the work for the day, this could be done earlier or later in the day. This made it very low stress and everyone in the lab was extremely friendly and fun to be around. The best part of the research was definitely at the end of data collection when we were able to compare the specific forces and see if the amniotic membrane supplementation was effective. However, the worst part was definitely the struggles that I had with a specific rats sample that took me three days to get 6 good fibers run. I also had a great time in the collaborative lab meeting that was help in August with the entire research team. This gave an amazing opportunity to share results from multiple aspects of the project and see how all of the data fits together.

Jeffrey Kepple is a senior at Gonzaga University in Spokane, WA. He is a 2017 Undergraduate Summer Research Fellow (UGSRF) doing research in Dr. Chris Mendias’ lab at the University of Michigan in Ann Arbor, MI.  After graduating, he plans on pursuing an MD/PhD.