Weekly Blog #1

Vocabulary to Know: 

Leukocyte/White blood cell- These cells circulate in the blood and are part of the immune system's response to injuries, damaging substances in the body, etc.

Blood Vessels/Venules- Both of these effectively mean the same thing for our purpose. This is where the red blood cells and most importantly, the leukocytes are traveling.

Endothelium- These layer of cells lines the inner walls of blood vessels.

Stuck Leukocyte- This cell is not moving because it is bound to the endothelium.

Rolling Leukocyte- This cell is still moving along the endothelium.

Ischemic Injury- These are injuries caused by low or lack of blood flow. The muscles do not receive oxygen. 

Summary: 

My Senior Project is to work at Dr. Jonathan Lindner's laboratory. He is one of the head cardiologists at OHSU and is allowing me to contribute to the team's current experiment. researching whether the inhibition of a protein called NLRP3 prevents white blood cells from causing ischemia reperfusion injuries. As part of my preparation, I analyzed peer-reviewed articles on the use of intravital microscopy. This is the technique we are using at the lab. An example of a case study I have read tracks alterations to the endothelial surface of microvessels using intravital microscopy to image the side effects of ponatinib, a tyrosine kinase inhibitor, often used to treat leukemia and cancerous cell growth in general.

My main job at the moment is to analyze footage from the intravital microscopy. Each video lasts about 10 seconds. What I see is one main vessel in the middle of the screen with tissue around it, and in addition to the red blood cells flowing quickly (creating blurred lines), I am focusing on small circles which are the leukocytes or white blood cells. First off I find the width of the vessel on the screen (or rather the diameter because blood vessels are 3D cylindrical shapes) and also the length of the segment of the vessel I will be examining. I then count the number of leukocytes that appear within that segment. There are 2 primary kinds of leukocytes: stuck leukocytes that do not move, and rolling leukocytes which can move either quickly or slowly based on their properties and how they interact with the endothelium, or the inner walls of the blood vessel. To normalize the number of leukocytes I find in each vessel, I divide the number of leukocytes by the surface area of the blood vessel (which I find using the surface area formula of a cylinder). This way I get leukocytes, or stuck leukocytes per mm^2 of endothelium.

The task is to find roughly 20 rolling leukocytes for each venule (blood vessel). There are more or less 10 of these venules for each mouse. There were at least 4-5 mice used in the experiment. Three of them were treated, and the other two are the control group. What they do is extract one of the leg muscles from each mouse, and cut off blood flow to the muscle causing an ischemic injury. Then, they allow blood flow to return to the leg and record how the leukocytes respond move differently in response. For each mouse there are a few videos of their venules previous to the ischemic injury, and then post-ischemia.

Why did I chose this project?

I plan on majoring in Biomedical Engineering in college and from there either getting a Master's in engineering, a PhD, or going to medical school. Since I already knew Dr. Lindner, this was a great opportunity to practice outreach and learn the value of connections. I feel this project is giving me a chance to get a first glance at the world I will soon be navigating on my own, in a new city, with new peers. I wanted to learn about the technology and procedures researchers use, and what kind of a work environment a research lab is.

Another priority for me choosing this project was also being challenged. The pandemic has certainly made me more mindful of how I spend my time efficiently getting work done or making memories with family and friends. I wanted to learn as much as possible and soak in everything for the month that we get for this project. I certainly feel challenged, just trying to understand the biology behind the data crunching I am doing. 

How did I pull this project together? 

I reached out to Dr. Lindner as early as possible as I was sure that, especially in the middle of the pandemic, his work being exposed to possible COVID-infected patients, was not only extremely busy but dangerous too. I first spoke with Dr. Lindner the first week of February. By the end of the week I had a solid Senior Project, the only problem would be if I had to do all of the work at home if things did not get better COVID-wise. I am incredibly lucky that I got my second dose of the Pfizer vaccine 12 days before starting at the lab so I was able to start my project on time without skipping a beat in person rather than having to work at home for the first chunk. 

Day 1 (Tuesday):

It had been a while since I read through the two background articles so I reread those. In addition, I switched articles with Emma Lindner who is also working at the lab. She read a separate article over the weekend about measuring the size of blood vessels and how that is relevant to the experiment. I explained my articles to her and she did the same before reading them on our own. This took a few minutes. We also figured out scheduling for the rest of the Senior project: Dr. Lindner will be available at OHSU on campus Monday, Wednesday, Friday. I may come in any day as I am mostly doing the same work in addition to some reading here and there. However, if I chose to come in on Tuesday or Thursday I need to coordinate with people at the lab so they can help me get in (people have to scan their IDs to get into most places so I can neither leave nor enter without someone else's help.

Day 2 (Wednesday): 

My dad dropped me off at OHSU at around 5:45 am on his way to work. I waited for roughly an hour and a half until 7:30 am outside Dr. Lindner's office. I wanted to start off the day as early as possible. Once he got there, we spent roughly the first 45 minutes at his office. He explained the experiment: how leukocytes move, what kind of data I will be working with, how to set it up on the Excel spreadsheet, etc. 

Then, we walked over to the lab in a different part of the campus. Once there I walked around watching as two men, one named Todd, and I did not catch the other man's name, both of them working on the mice. One was performing some kind of surgery before bringing that mouse to Todd who then performed the intravital microscopy and the ischemic injury. I also met Koya, a kind Japanese scientist that welcomed me to the lab. I also met Matt Meuller, the most tech-savvy guy of the bunch that Dr. Lindner has been trying to kick out of the lab so he goes to grad school but as it seems, he is jus having too much fun working at the lab. As it turns out, there was also another guy named Matthew (I don't remember if they also referred to him as Matt). So up until now, we have Dr. Lindner, Koya, Matt, Matthew, Todd, mystery man (who is by the way also really sweet), and a graduate student named The Anh Nguyen whom I will be working with.

We then proceeded to spend the next two hours I would say trying to figure out how to measure distance (in mm) on the videos after callibrating the software so that it knows the ratio of pixels to microns. The goal was to draw a line on the video and then find the length of that line in microns. We needed the full brain power of Dr. Lindner, Koya, and Matt to figure this out. At that point it was roughly 10:00 am. I helped Dr. Lindner record the data for the stuck leukocytes for two of the three treated mice. By 11:00 am I had caught on and worked for the next hour and a half until 12:30 pm gathering data of the venule 2 footage, for the first treated mouse. By then I was exhausted and headed home to relax for the rest of the day.

 I had actually not eaten breakfast or lunch and I was starving so that was the first thing I did when I got home. It was an exhausting day but so interesting and overwhelming in the best possible way. 

Day 3 (Thursday): 

By now I was a total pro. Dr. Lindner told me to come in whenever I wanted and coordinate with Matt so that he can let me in and help me out in case I needed anything. I arrived at the lab around 9:45 am. I caught up on my Senior Project Blog until 11:00 am. I then spent the next three hours until 2:00 pm tracking leukocytes on my screen. 

Day 4 (Friday): 

I arrive around 5:30 am again to get some work done. Then I sat in on one of the conferences in the morning with fellows of the cardiology department. Two of the fellows shared each a physiology article from two or three decades ago outlining basic understanding of the human body. These were fascinating studies. One of them talked about whether the heart will adjust, and lower the effort it is making to pump blood if there is less blood (in other words, will it conserve energy?). 

3 Goals:

1. I want to learn about the process of getting funding for research, fining a lab space, putting together a team, etc. all of the logistics.

2. I want to learn the ins and outs of the experiment that Dr. Lindner and his team are carrying out. 

3. I would lastly like to learn about what steps Dr. Lindner and his team take to treat the mice humanely and whether similar experiments could be run either without taking the animals' lives.

In learning about these three aspects of research, I want to start to have some idea about what I would like to study in college within biomedical engineering, what kind of research I would like to do, and what is the right path for me out of my undergrad: PhD? masters in engineering? med school?