Tag Archives: physiology

PoWeRful mice and the effect of satellite cell depletion
Alec Dupont
Junior, biomedical science major
Auburn University

My Research Project

My project involved examining the adaptation of skeletal muscle to resistance exercise in mice that had been depleted of muscle stem cells (satellite cells). Generally, muscle growth is accompanied by an increase in protein synthesis and the differentiation of satellite cells into muscle nuclei. During this project, we examined if growth happens without the addition of satellite cells into muscle. As certain clinical populations have reduced satellite cell content and muscle mass, our project aimed to provide insights into how muscles respond to a growth stimulus with the loss of this cell population.

We used Progressive Weighted Wheel running (PoWeR) as a model for resistance exercise. PoWeR involves voluntary running activity of the mice in weighted running wheels. The weight placed on the running wheel is gradually increased over the course of four to eight weeks, overloading the musculature and causing a growth response called muscle hypertrophy. Using a genetic mouse model that allowed for the selective depletion of satellite cells, we compared sedentary- and resistance-exercised mice in groups of satellite cell-replete (vehicle treated) and -depleted (tamoxifen treated) mice. We compared muscle hypertrophy and other physiological adaptations between groups to determine the effects of satellite cell depletion. At the completion of this project, we hoped to gain a further understanding of the role satellite cells play in muscle growth.

Realities of Research

My main focus for the summer was using muscle tissue from the PoWeR mice, and making it possible to obtain data and useful information. I accomplished this through immunohistochemistry, a laboratory technique where we cut cross sections of the muscle and stain them for proteins of interest. This staining allowed us to visualize the sections under the microscope, image them and quantify the images using different forms of software. This technique presented certain challenges because the tissue must be carefully prepared and stored to prevent degradation. Poor quality tissue introduced variability outside of what is normal to the mice models. For example, having to overcome challenges and work to optimize a stain meant visualizing newly formed RNA in muscle nuclei. The stain can appear too dull and the quality would not be high enough to draw conclusions unless the procedure was optimized. Overcoming these challenges provided stunning images and reliable data. We found that although satellite cells were not absolutely required for muscle growth in response to weighted wheel running, there was a decrease in growth in the satellite cell depleted mice.

Life as a Scientist

The day-to-day life of a research scientist presented me with a constantly changing experience that was more engaging than the traditional classroom setting. There was always a new aspect of the project to investigate. It was incredibly satisfying to see your work come together in data that tell a cohesive story. The process of getting there was occasionally tedious though. For example, we’d normalize our data to the number of fibers in the muscle cross section and when the software couldn’t count for us, we were forced to count by hand. When the sections were between 600 and 800 fibers in a study with 48 mice, that part of research tended to drag. But that was only a minor inconvenience to a necessary bump in the road towards a satisfying research project.

Alec Dupont is a junior at Auburn University in Auburn, Alabama, studying biomedical science. He is a 2019 Undergraduate Summer Research Fellow (UGSRF) working under Dr. Charlotte Peterson at the Center for Muscle Biology at the University of Kentucky in Lexington. Alec’s work is funded by the American Physiological Society’s UGSRF program and a grant from the National Institute of Health to Dr. Charlotte Peterson and Dr. John McCarthy (AR060701).

A Summer Study: Respiratory Rehabilitation After Spinal Cord Injury
Amari Thomas
Senior, Biology
University of Florida
2019 STRIDE Fellow

My Research Project

The human body central nervous system.

Because the central nervous system is in control of every process taking place within the body, an injury to this system can be detrimental and sometimes fatal. Injuries to the cervical region of our spinal cord can be extremely difficult because they often lead to breathing impairment. The phrenic motor nucleus in this region innervates our diaphragm, which controls inhalation by creating a negative pressure ventilation system.

It has been shown that acute intermittent levels of low oxygen help to address the concern for the functional recovery of breathing after injury. This occurs because the phrenic motor nucleus elicits neuroplasticity. A key protein, phosphorylated-ERK (p-ERK), is involved mechanistically in the phrenic motor nuclei response to varying levels of low oxygen.

P-ERK’s expression can be analyzed through epifluorescent microscopy. The cervical spinal cord tissues were harvested from rodents and stained using inmunoflouresence, – a procedure that stains the tissues in a way that allows them to emit certain colors when viewed on a microscope. We injected cholera toxin B between the pleural cavity in the outer layers of the rodents’ lungs before injury, which allowed for selective localization of phrenic neurons. We imaged this tissue to assess different expression patterns of p-ERK after spinal injury and varying levels of intermittent hypoxia.

Once we analyzed the expression of p-ERK in phrenic motor neurons after spinal injury and intermittent hypoxia we were able to develop a better understanding of intermittent hypoxia and its elicited plasticity after spinal injury. This research will guide therapeutic strategies for improving breathing in people with spinal injury.

Life as a Scientist

Using rat models as a method for testing before human clinical trials.

My experience as a scientist this summer opened my eyes to the realities that occur behind the scenes of groundbreaking research. For example, I always believed clinical trials to be amazing advancements in research, but never truly understood all of the experiments that take place before humans are even brought into the picture. The work done in our lab on rats propose a model for human experimentation. This opportunity has also made me realize that things may not always go exactly as planned the first time around and that is perfectly okay. Often, these trials and errors allow us to learn more about the research we are doing in order to propose different hypotheses or use alternate methods. There is no right or wrong when it comes to research because it is a learning and growing experience.

Acknowledgements

Elisa Gonzalez-Rothi, DPT, PhD, Research Assistant Professor, University of Florida Department of Physical Therapy

Gordon S. Mitchell, PhD, Professor of Physical Therapy, University of Florida Department of Physical Therapy

Latoya Allen, PhD, University of Florida Department of Neuroscience

Marissa Ciesla, PhD, University of Florida Department of Neuroscience

Amari Thomas is a first-generation college student majoring in biology at the University of Florida in Gainesville. She was born and raised in Miami Gardens, Florida, where access to research labs and quality educational resources are minimal. Due to her academic success in grade-school and extracurricular involvement, Amari was accepted into one of the top universities in the country for her undergraduate education. She has continued to thrive in her undergraduate career by gaining dean’s list awards for academics, mentorship positions and an outstanding fellowship from the American Physiological Society. By working in a research lab, Amari has expanded her career options and strengthened her knowledge of the human body and its many processes. She hopes to obtain a medical license after graduating and plans to apply the knowledge learned in the research lab. Amari is a 2019 Short-Term Research Education Program to Increase Diversity in Health-Related Research (STRIDE) Fellow in the lab of Dr. Elisa Gonzalez-Rothi at the University of Florida in Gainesville. Amari’s fellowship is funded by the National Heart, Lung and Blood Institute (NHLBI; R25 HL115473-01).

Multisensory Control of Blood Pressure

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

  1. Yates, B. J., P. S. Bolton, and V. G. Macefield. 2014. Vestibulo‐sympathetic responses. Compr. Physiol. 4:851–887.
  2. 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
  3. 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.