November | 2018 | APS Undergraduate Researcher

When we go from a lying down position to standing up, our blood tends to pool in our legs and has to fight against the force of gravity to travel our brain. Overcoming the force of gravity is the greatest challenge the cardiovascular system faces. Luckily for us, our body is able to detect when such changes are to occur and can effectively change blood pressure to compensate for the change in posture. The body has many different sensory inputs that contribute to homeostatic regulation of blood pressure during these postural changes. The vestibular system, baroreceptors, chemoreceptors, and higher order brain centers (hypothalamus and cerebral cortex) are all examples of the body’s many sensory inputs (Yates et al. 2014). If these homeostatic responses perform inadequately, then conditions such as orthostatic hypotension can be the result (Yates et al. 2017). Orthostatic hypotension results from a drop in blood pressure due to a change in posture, where insufficient perfusion of blood with the brain occurs. My research project this summer focused on the cardiovascular responses that are necessary to maintain normal biological homeostasis during anticipated imposed postural changes. Homeostasis is defined as the tendency of biological systems to maintain relatively constant conditions in the internal environment while continuously interacting with and adjusting to change originating within or outside of the system (The Free Dictionary, n.d.).

In this study, my lab investigated the activity of neurons in the RVLM (rostral ventrolateral medulla), located in the brainstem, in awake adult female cats. There has been significant research to support the claim that neurons in the RVLM play in important role in blood pressure regulation in both animals and humans (Yates et al. 2017). RVLM neurons are necessary for cardiovascular responses to maintain homeostasis during postural movements. The neurons in the RVLM were identified by their changes in firing rate correlated with the cardiac cycle and their location. The experiments in the lab involved recording the activity of neurons in the RVLM during imposed 40º head-up tilts that were preceded by a light cue. The light cue occurred 10 seconds before the 40º tilt, and the heart rate and firing rate of neurons were determined for 5 second time periods before and during the tilts.

Our hypothesis is that in neurons that exemplify cardiac related activity there is an increase in neuronal activity prior to the onset of the 40º imposed tilt, in the time interval after the light stimulus as compared to before the light. This would indicate that when the animal is able to anticipate when the change in posture is about to occur, the animal’s cardiovascular response system will increase heart rate and blood pressure to prepare for the postural change. This would be an example of a feedforward mechanism rather than a feedback. In a feedback mechanism, the animal would have already undergone the change in posture and body positon in space before the cardiovascular response system could compensate heart rate and blood pressure. This research project will ultimately further our knowledge of cardiovascular homeostasis and may lead to new treatment options for orthostatic hypotension and also a treatment for cognitive mechanisms that can cause changes in blood pressure such as stress and anxiety.

Figure 1: An example of the data that was collected and analyzed during one of the tilt table experiments. The x-axis shows the change in time as the table went from a nose down position, to a 40º head-up tilt. The neuronal firing was isolated along with the heart rate through the use of an EKG. The sun cartoon indicates when the light stimulus was shown with the five second time intervals appropriately labeled before and during the head-up tilt. Credit: Dr. Bill Yates’ Lab, UPMC Department of Otolaryngology.

Realities of Research

I was extremely grateful to take part in this research opportunity this summer as it really allowed me to grow as an undergraduate researcher. The reality of research is that you get out of it what you put in. If you’re motivated and passionate about what you are doing, then I believe that anyone will be satisfied with the outcome of their experience. I became enamored with learning about physiology after completing Dr. Yates Honors Human Physiology class. After completing his course, I was eager to investigate what research was like on the physiological spectrum. Over a course of 10 weeks, I completed dozens of experiments and analyzed large amounts of data. We performed single-unit in vivo recordings using electrophysiological techniques and procedures. When experiments ran to completion and a respectable amount of usable data was collected, the day was considered successful. Another reality of research is that not every experiment is going to go as planned. There were experiments where either the animal wasn’t cooperating on the table, an electrode bent, or we just simply couldn’t isolate any good neurons to record from. When problems like these would occur, we would simply end the experiment and try again the next day. Because there are a great deal of experiments left to perform and more data analysis to complete, there are no final results or conclusions yet to be made. However, we do predict that the firing rate of the cardiac-related neurons will increase after the light stimulus is given, prior to the onset of the tilt.

Figure 2: The tilt table that is used in all of the single-unit in vivo recordings. During an actual experiment, the curtains would be drawn and the lights would be dimmed to allow for the light stimulus to appropriately work. The machines in the back are used to find and isolate a neuron in the RVLM and to change the position of the table. Credit: Dr. Bill Yates’ Lab, UPMC Department of Otolaryngology.

Life of a Scientist

I sincerely enjoyed my daily routine as a scientist in the Yates lab. I was able to set my own schedule and I developed meaningful friendships with everyone I worked with. It was a very-low stress working environment and most days I would follow the same routine. A huge part of my project this year was analyzing the data collected from the experiments. When I wasn’t running any experiments I would be on the computer analyzing large amounts of data. Analyzing data is something that a lot of undergraduate students have trouble with, mostly because it takes a lot of time to learn and pick up. I was very surprised by how quickly I was able to pick up the skill and it allowed the lab to make huge amounts of progress throughout the summer. My fondest memory of my time in the lab didn’t actually happen in the lab. On a Friday afternoon after work, everyone in the lab went out to a Pirates game at PNC Park in Pittsburgh. It was my first one and it was really fun to see everyone outside of the lab doing something together. Overall, I had a great summer and I’m very thankful that APS gave me the opportunity to see what full-time research was actually like. This summer experience was very rewarding and has motivated me to pursue my continued interest in biomedical research.

References

Yates, B. J., P. S. Bolton, and V. G. Macefield. 2014. Vestibulo‐sympathetic responses. Compr. Physiol. 4:851–887.
Patel, N.M., Baker, E.A.G., Wittman, S.R., Engstrom, I.C., Bourdages, G.H., McCall, A.A., Miller, D.M. and Yates, B.J. Cardiovascular adjustments during anticipated postural changes. Physiol. Rep. 6(1), e13554, 2018
TheFreeDictionary.com. (2018). Homeostasis (Biology). [online] Available at: https://medical-dictionary.thefreedictionary.com/Homeostasis+(Biology)

John Bielanin is a rising senior majoring in Neuroscience and minoring in Chemistry, with a certificate in Conceptual Foundations of Medicine at the University of Pittsburgh in Pittsburgh, PA. He is a 2018 Undergraduate Summer Research Fellow working in Dr. Bill Yates’ lab in UPMC’s Department of Otolaryngology at the University of Pittsburgh. John’s Undergraduate Summer Research Fellowship (UGSRF) is funded by the American Physiological Society. Outside of school and work, John enjoys reading, writing music, and spending time outdoors. After graduation, John plans on applying to medical school while continuing to pursue research opportunities in his gap year.

Preeclampsia, a hypertensive disorder in pregnancy, affects more than 6 million pregnancies per year worldwide. It is a dangerous condition during pregnancy which involves high blood pressure, proteinuria, and swelling. The Santillan lab has shown that single dose early of BMTZ early in pregnancy will reverse late pregnancy hypertension and proteinuria (1). The molecular mechanism by which this reversal occurs is unclear. One potential pathway involves serum/glucocorticoid regulated kinase 1 (SGK1), a serine/threonine kinase stimulated by corticosteroids. SGK1 dysregulation and human genetic variants in SGK1 have been associated with hypertension. The overall goal of this study is to determine whether SGK1 and its regulation play a role in preeclampsia. If so, BMTZ has the potential to prevent preeclampsia in humans. Because mir-365 has been shown to decrease SGK-1 expression in human placentas from pregnancies with poor placentation, I will examine the effect of vasopressin and SGK1 in placental cells on mir-365 expression. In addition, it will be determined if placental mir-365a-3p is differentially expressed in human preeclampsia. This project is significant because it may help to determine how BMTZ protects from preeclampsia and whether BMTZ could be useful in humans.

Research in the lab can be very stressful. Things may not go as well as expected and troubleshooting is a process. Regardless, I had the opportunity to learn many new techniques that would help me in the future. I was surprised how research is comprised of so many different aspects. A little difference in one experiment may change the whole outcome. I learned a great set of skills like how to maintain a cell culture, perform an ELISA, BCA, and extract RNA. It took a while for me to start up on my experiments because I had to research some more background information to ensure I knew what I was doing. My experiments went smoothly, but it was later found that the drug I was using to treat the cells was not working in our mouse model; therefore, it may not be working with my cells as well. My project was put to a halt to first determine if the drug was correctly performing. The drug was aliquoted about a year ago and may have degraded. I would have to wait in order to determine whether I was able to continue or to start over. In the meantime, I worked with my mentor with small projects and learned useful techniques. Additionally, I worked on the second portion of my project involving whole placental tissues. The tissues were RNA prepped and analyzed via qPCR. The results showed that there was a significant difference with p-value of 0.016. This makes sense because Xu found that miR-365 negatively regulates IL-6 and it, in turn, is transcriptionally regulated by Sp1 and NF-κB. (2) So, transcriptional down-regulation of miR-365 should result in increased IL-6. This was interesting to hear, but we cell culture was needed to determine this and it was on standstill.

There were some busy days and other days there was a lot of down time. For example, one day there may be multiple tests to complete in a day, other days an experiment would consist of wait time. The most surprising part of participating in the lab is that I realized that a lot of the down time is used to write papers or grants. Research involves a great deal of writing to express the study to the public eye and document previous studies to help ongoing studies. I am appreciative of researchers because without those papers I would not have been able to understand my study without background information. Most days were very stressful trying to balance all of the work and trying to understand why a certain mechanism happened. My least favorite part during my time in the lab was working so hard on an experiment and in the end, not having it work out. The best part was working along with my mentor to learn new techniques and tests. I’m also glad that people around the lab worked well with one another and that they would take the time to reach out and teach me.

References:

Santillan, M., Santillan, D., Scroggins, S., Min, J., Sandgren, J., Pearson, N., Leslie, K., Hunter, S., Zamba, G., Gibson-Corley, K. and Grobe, J. (2014). Vasopressin in Preeclampsia: A Novel Very Early Human Pregnancy Biomarker and Clinically Relevant Mouse Model. Hypertension, 64(4), pp.852-859.
Xu et al. miR-365, a Novel Negative Regulator of Interleukin-6 Gene Expression, Is Cooperateively Regulated by Sp1 and NF-κB. Journal of Biochemistry 286: 21401-21412, 2011

Carolyn Lo is a junior majoring in Human Physiology and Biochemistry at the University of Iowa in Iowa City, Iowa. She is a 2018 Short-Term Research Education Program to Increase Diversity in Health-Related Research (STRIDE) Fellow working with Dr. Mark Santillan at the Carver College of Medicine in Iowa City, IA. Carolyn’s fellowship is funded by the APS and a grant from the National Heart, Lung and Blood Institute (NHLBI) (Grant #1 R25 HL115473-01). After graduation, Carolyn plans to pursue a doctorate degree in medicine.