Tag Archives: spinal cord injury

Cycle Training promotes bone growth following Spinal Cord Injury
Jayachandra Kura
Junior, Applied Physiology and Kinesiology
University of Florida
2019 UGSRF Fellow

My Research Project

Figure 1. Transverse view of long bone with red ROI

This past summer, I worked in Dr. Joshua F. Yarrow’s research lab at the Malcom Randall Department of Veteran Affairs Medical Center. Dr. Yarrow’s lab explores the effectiveness of pharmacologic and exercise treatments following spinal cord injury (SCI). For the specific SCI we studied, the posterior end of the 9th thoracic vertebrae was surgically removed, exposing the spinal cord underneath. A machine delivered an impact causing hindlimb paralysis. My research  used Sprague-Dawley rats that were given either a 1) SCI, 2) surgical control (SHAM), 3) SCI + Bodyweight Supported Treadmill Training, or 4) SCI + Passive Bicycle Training. We scanned the distal femurs at baseline, two weeks and four weeks after SCI using a micro tomography (microCT) scanner.

In order to observe the effect of each treatment on the spongy cancellous bone, a technician would individually draw a region of interest (ROI) in the transverse view of the femur (Fig. 1) to include the internal trabeculae while excluding both the growth plate and solid cortical bone. However, repeating this on more than 100 slices for every sample at every time point is very time intensive. Instead, I worked to adapt a registration procedure for the spinal cord injury model. The registration was created by using two scans at different time points are aligning them in 3D. An ROI was created at baseline and then applied to the two-week and four-week scans, reducing the amount of labor required. I then compared the data from registered images to data from nonregistered images. I also helped to develop a script that allowed the computer to automatically draw the ROI with minimal manual correction, which further improved efficiency.

Realities of Research

Figure 2. Spinal Cord Injury Model. However, instead of a contusion by weight drop, there is a machine performing the impact.

My introduction into research has definitely been equal parts trying and gratifying—trying in the sense that every solution I created seemed to raise a host of other questions that needed to be addressed. I remember when I finally figured out how to register two different time point images, but I then needed to decide what size volume of interest (VOI) to use so the computer knew which landmarks to use to align the two different bones. Intuitively, using a large VOI should provide more datapoints for the computer to use. Doing so caused the solid cortical borders to be well-aligned, but the internal structures weren’t. Repeating this with multiple samples yielded the same results, which suggested that, in bone remodeling, an individual bony landmark’s relative position to the cortical border changes with time. However, using a small VOI caused poor alignment of the two images. This seemed counterintuitive, so when I looked over previous scans of SCI samples, I observed a trend of severe bone loss occurring below the injury site. The registration procedures outlined in the literature couldn’t directly translate to a SCI model as those outlined procedures required clear internal bony landmarks. Without these data points, the automatic registration software couldn’t produce an accurate alignment.

In this seemingly never-ending cycle of forming new ideas only to eliminate them later on, I didn’t come any closer to developing a script, but I did develop a lot of patience and perseverance. I found research to be inherently challenging, but the setbacks I encountered only made me grow as a person and researcher, and ultimately, made the end result of creating a “mostly” functioning protocol all that more rewarding. I’ve also come to realize that there is never a true “end” in research as there arealways things that can be improved or new questions that can be asked. This opportunity for continual growth was really exciting and intrinsically motivating.

Life as a Scientist

Compared to my past work experiences, research has by far been the most enjoyable. Never did I have a bout of the “Sunday scaries,” where I was enveloped with the existential dread of going to work the following day. In contrast, my work environment was low-stress and was dictated entirely by my own drive and will to work. The lab was filled with diverse, interesting individuals and I enjoyed the conversations I had and the relationships I formed with my labmates. Although there was always monotonous data entry, most of the work I did within the lab was challenging and fun. I always felt the work I was doing was meaningful.

I recall a conversation I had with my labmate who’d recently graduated: I had jokingly asked what it was like not having class. He laughed and said, “I spent the last four years—every fall, spring and summer semester—taking classes and working here at the hospital. If you think about it, when you’re an undergraduate, you’re basically working 70+ hours a week with all the stuff you do, so you really never have to think about anything except for school. Now that I’ve graduated and work 40 hours a week here in the lab, my work ends when I leave. But I remember going home and sitting on my couch not knowing what to do with myself, thinking, ‘Man, time to find some hobbies.’” Being out of school, if only briefly, allowed me to finally begin to appreciate this. Now sitting on my own couch trying to find things to do, I’ve found this freedom to be exciting and paralyzing.  I definitely feel that the physician/scientist career path is like a pipeline and there’s constant pressure to continue moving towards the end. To be honest, I haven’t put much thought into the adult I want to be outside of my career or really explored the things I find fulfilling. I’m just thankful for the opportunity to have had these experiences, both in and out of the lab, and believe that this summer was largely beneficial for my growth not only as a researcher, but also as a person.

References:

L Arsuaga, J & Villaverde, Valentín & Quam, Rolf & Martínez, I & M Carretero, J & Lorenzo, Carlos & Gracia, Ana. (2013). Arsuaga et al. 2007.

“Establishment of a Rat Model of Spinal Cord Injury (SCI).” Neural Regeneration Research, www.nrronline.org/viewimage.asp?img=NeuralRegenRes_2016_11_12_2004_197145_f1.jpg.

Jayachandra Kura is a junior majoring in applied physiology and kinesiology and minoring in Japanese at the University of Florida in Gainesville. He is a 2019 American Physiology Society Undergraduate Summer Research Fellow (USGSRF) working in Dr. Joshua F. Yarrow’s lab at the North Florida/South Georgia Medical Center in Gainesville, Fla. Jayachandra’s fellowship is funded by the American Physiological Society and the Department of Veterans Affairs. After graduation, Jayachandra plans to pursue a career as a physician scientist.

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.
A Glimpse Into My Fight Towards Finding a Safe and Effective Way to Combat Respiratory Dysfunction

My research project this summer focuses on the development of rehabilitative strategies used to combat respiratory dysfunction (the inability to breathe) in patients suffering from spinal cord injuries. Following a spinal cord injury, brainstem projections traveling down the spinal cord are severed, ultimately leading to paralysis of respiratory muscles such as the diaphragm. Consequently, breathing impairments are the primary cause of death for spinally-injured patients. This summer project focuses on a rehabilitative therapy known as intermittent hypoxia (IH) which involves brief and repeated exposures to low oxygen levels over a period of time. When given in low doses (>15 episodes/day), intermittent hypoxia can improve leg strength, walking function, and respiratory function in spinally injured rats and humans. On the other hand, high doses of intermittent hypoxia can elicit spinal inflammation and other pathological consequences. My study seeks to determine whether activation of a certain protein kinase that acts as a marker of inflammation, p38 MAP kinase, is increased in all dose protocols of intermittent hypoxia or just high dose intermittent hypoxia. Assessing the impact of p38 MAP kinase phosphorylation/activation is crucial for the development of safe and effective methods to restore respiratory function in individuals with a spinal cord injury. Currently my research subjects are rats; however, once our lab can ensure that low dose intermittent hypoxia is safe and non-harmful to humans, through my project and many others currently being conducted, we can transition our treatments from animals to humans.

What was it like doing research in a lab?

My research experience this summer was an eye-opening experience that, for the first time in my professional career, allowed me to devote all my time to the research I am passionate about. Conducting research in the lab alongside other undergraduate and graduate students, and post-doctoral fellows, was nerve-wracking yet very rewarding. When I first began my project, part of me was afraid to attempt tasks and techniques that were new to me for fear of ruining valuable data. However, to my surprise everyone in the lab was eager to help me in any way they could even if they were occupied with their own projects. I quickly learned that I would have to develop several techniques for my project to be a more independent and productive researcher. These techniques include immunohistochemistry (IHC) staining and imaging. Thus far, my experiment has been working, with all CTB injections, exposure treatments, perfusions, harvesting, and tissue sectioning going successfully. An unexpected mishap that occurred involved the immunohistochemistry staining. During our first IHC staining trial, we stained only a few of the tissues and had difficulty viewing the markers that were stained for when imaging. This was a result of having used an antibody at a wavelength of light that wasn’t appearing on the microscope. After reviewing literature and past protocols we decided on a different antibody that would produce better images. Although unfortunate, this setback demonstrated that while conducting research several things can go wrong but can just as easily be fixed. As for results, my project is still under the works as I have just reached the imaging phase.

I have found my experience as a scientist this summer to be both rewarding and stimulating. Every day when I come into lab, I am eager to jump into my project and engage in conversation with other researchers about the impact all of our research could one day have. My days consist of running experiments, performing animal care, cutting tissue, and whenever I have down time, reading papers. The best part of my research experience is collaborating with other members in the lab. I enjoy listening to the advice and thoughts of the other researchers because it gives me other perspectives on things. The worst part about being a research scientist is probably accepting mistakes and figuring out how to solve the problems. I found working as part of the team to be an educational experience that helped me improve my communication and leadership skills.

 

Juliet Santiago is a junior majoring in Microbiology and Cell Science at the University of Florida in Gainesville, FL. She is a 2017 Short-Term Research Education Program to Increase Diversity in Health-Related Research (STRIDE) Fellow working in Dr. Gordon Mitchell’s lab at the University of Florida in Gainesville, Florida. Juliet’s fellowship is funded by the APS and a grant from the National Heart, Lung and Blood Institute (Grant #1 R25 HL115473-01). After graduation, Juliet plans to pursue a career as a biomedical scientist in industry or government.