Week 7: University of Chicago Medical Center

August 6th, 2012

Jordan Kemme ’13, Becker Fellow in Neuroscience

I missed my last blog post because I wanted to be able to share all of the results and conclusions I have been able to make after my experiment was complete. The past two weeks have been very exciting. We observed successful cellular death within our cultures which allowed us to carry out our experiment. We used lectin staining to determine that cultures we treated with clodronate liposomes had reduced microglial populations compared to control cultures treated with PBS liposomes that have no cytotoxic effect. Finally, we determined the spreading depression susceptibility of our experimental and control cultures. By stimulating one region of a hippocampal slice culture and recording the field potential in another region we were able to determine what stimulatory current was necessary to induce a spreading depression. The current that induces spreading depression was recorded as the threshold current. We ultimately found that hippocampal slice cultures with depleted microglial populations have significantly decreased spreading depression susceptibility when compared to hippocampal slice cultures with normal microglial populations. These preliminary results indicate that microglial cells are important and possibly essential for spreading depression induction and subsequently migraine headache.

Our data are preliminary because the majority of our cultures were infected during the treatment period leaving only 3 experimental slices and 6 control slices that were uninfected to work with. However, we have set up more cultures and began treatment again because our preliminary results were significant as well as reassuring. We have been toying with different ways to improve our technique that may allow us to eliminate higher amounts of microglia in addition to better observe healthy hippocampal slice culture electrophysiology. We are putting these ideas into practice in the last few days I have here. It is very exciting to know that I have discovered a plausible cause for migraine and even more so a pharmacological target that may be used in the future to treat the 30 million Americans that suffer from migraine each year.

As my research at the University of Chicago is coming to an end I am preparing numerous presentations and papers. I will be finalizing a detailed scientific paper describing the process I took this summer to explore microglial involvement in spreading depression and migraine. I have prepared a power point presentation which I will be delivering to numerous colleagues and University personnel on August 8th. The presentation has a 10 minute time limit which scared me initially but as I practiced and revised my presentation the 10 minute time frame has become more reasonable. I have also completed a poster which I will be presenting August 8th at the University of Chicago’s research symposium. I am confident in my ability to describe my project as well as the underlying themes it pertains to. That said, I am very excited for the presentations I will be giving within the next few days. However, what has me most animated is the process that will begin this fall when our lab will begin the publishing process for the work we did this summer in which I will be the second author!

This summer was more than an amazing experience. I was offered the opportunity to work in a premier laboratory for the first time in my undergraduate career. I was terrified and intimidated at first but after several weeks I found that with persistence I would accomplish my goals. I learned to confront failed attempts happily because in nearly every instance of failure this summer the chance to learn something new about my project was revealed. Our teachers aren’t kidding when they say we learn the most from our mistakes. I’m both relieved and upset that the summer is coming to an end as it was both a good learning and social experience. All in all I’d have to say there were four major lessons I learned this summer: (1) Show up to lab every day–yes, Saturday is one of those days, (2) drink your coffee, (3) do the reading, and (4) you get out what you put in.

Week 5: University of Chicago Medical Center

July 20th, 2012

Jordan Kemme ’13, Becker Fellow in Neuroscience

I don’t have a huge update this week since we have been struggling with the same problem for about two weeks now. We are not sure if the method we are using to selectively eliminate microglia from our slice cultures is effective. We use SYTOX media every other day to determine the relative amount of cell death in the cultures. SYTOX is an intercalating agent which will bind to free-floating DNA after it has been released from a dead cell due to the degradation of a dead cell’s membrane. Once SYTOX binds to DNA it fluoresces and we measure the intensity and compare the relative intensity of fluorescence to control slice cultures. I believe one problem with this method is that after DNA is released from a dead cell nucleus it is usually degraded by DNAse activity. To me, this means that our cultures could be experiencing large amounts of cell death however, because DNA is degraded, SYTOX staining may indicate less cell death than what actually occurred. The only way to truly know if we have depleted the microglia population is to use lectin staining. Lectins are proteins that specifically bind to glycoproteins found in certain cell membranes. Lectin specificity allows a researcher to identify the relative population of a certain cell type within a culture. We use lectin to stain for microglia. If we find that microglia populations are significantly less in cultures we have been treating compared to controls we will know that our method is effective. Hopefully I will be able to look at a slide I made a few days ago using lectin staining in order to see how effective our method was. Lectin staining may be useful however, and in order to perform it the cultures have to be fixed which renders them unable to perform anymore physiological properties. In our case, we need our cultures alive to perform our spreading depression experiment so we have to trust that the right cells are dying using the SYTOX method before we can confirm that microglia cells were not present.

During the long incubation periods I have spent my time looking up all the literature that pertains to our research topics. Dr. Kraig is planning on writing a paper on our findings so we need to be sure that we cite every single paper that has used methods similar to ours, even if we didn’t use the paper, in order to avoid fraudulent accusations. Citing papers is not a new idea for me, however, I was never familiar with how serious the repercussions could be. These new ideas are giving me a greater understanding of the politics behind publishing a scientific article. In the meantime I am going to scour the web in order to find and cite all the appropriate articles. Keep your fingers crossed for me!

Week 4: University of Chicago Medical Center

July 10th, 2012

Jordan Kemme ’13, Becker Fellow in Neuroscience

After a month working in Dr. Richard Kraig’s lab I am finally starting to make some progress with my experiment. My current goal has been to show that the method we want to utilize only kills microglia cells while leaving the rest of the hippocampal slice culture in tact. In our most recent cytotoxicity experiment the control indicates that the method in which we want to deliver our drug specifically to microglia is not toxic to the entirety of our slice cultures meaning that it is a practical option for us. However, the drug itself turns out to be acidic which presents a problem itself. The acidic nature of the drug we are using causes of the pH of the culture media to fall below the physiological pH at which cells are viable.  The decreased pH may cause excessive neuronal death which is not favorable for our experiment.  We will most likely be able to fix this by titrating our drug in an aqueous solution to decrease its acidity which will inevitably bring the pH of the media closer to physiological pH. Believe it or not, I don’t actually mind these road blocks. The problems that we incur present us with the opportunity to explore literature for solutions. While looking for solutions we almost always learn something new about our procedure or the underlying theories behind our experiment. I enjoy using the research tactics that countless Cornell courses have taught me to answer the questions my research poses.

Today I went to watch one of the Ph.D. students I work with give a presentation on her doctoral work to several students and faculty. It was good to watch her talk since I will be presenting on similar topics at the end of my fellowship to a similar audience. I was able to see what kind of format I should use to set up my presentation and what kind of questions I should anticipate from the audience. Watching several of these presentations made me realize how lucky I am to of already had the chance to make several presentations in my courses at Cornell. My past experiences will definitely contribute to the methods I will use to present my findings to the rest of the students in my program during our symposium on August 8th.

Week 3: University of Chicago Medical Center

July 1st, 2012

Jordan Kemme ’13, Becker Fellow in Neuroscience

I have completed roughly three weeks here at the University of Chicago working in Richard Kraig’s lab. My lab work has been progressing nicely and I am beginning to feel more comfortable with the protocol for my experiment. We are trying to prove that microglial cells are responsible for the physiological phenomenon known as cortical spreading depression (CSD), a wave of electrical inactivity spreading through the cerebral cortex of the brain that is responsible for migraine headache and aura. My task is to eliminate all microglial cells from a hippocampal slice culture in order to determine if CSD is dependent on microglial cells. The task of eliminating microglia cells has proven to be difficult. I have spent the majority of my time reading articles as well as a neuroscience text book to learn more about the nervous system in order to design the best protocol. I am very close to completing my task which means that I will soon be moving forwards in my experiment.

When I am outside the lab I have other tasks to complete required by both my lab director and the program I am participating in. I am required to produce a paper by the end of the summer, however, the program directors have developed a method to insure we don’t procrastinate and write our paper within the last few days of the program. Each week we write a section of our paper (references, hypothesis, intro, etc.) and upload it to an online database that contains a chart of all the students in my program. If we miss the deadline, the chart displays a red box under our name and all the students in the program are able to see it. In order to avoid humiliation, we have learned to turn in our materials on time; I will admit that it is good incentive to get my work done early. Furthermore, it has been helpful completing my intro so early on in the experimentation process because I feel like I have a deep, more solidified understanding of my topic in both the broad and narrow sense.

In addition to writing a paper and reading materials for my lab we also have weekly “cluster group” meetings in which we present our work and progression in lab to the other students in the program and several Ph.D./M.D.’s. We don’t use powerpoint so I have become more comfortable speaking without visual cues and answering any questions the audience has. The doctor’s will to drill us on our underlying knowledge of our projects is comparable to the many comments I have received presenting in my biology classes at Cornell. The past two weeks have allowed me to improve upon my presentation skills and learn how to work more independently in lab. My lab director has given me more responsibility as I learn more about the protocol which has allowed me to become more of an independent thinker. As independent as I may feel, however, I benefit most from the discussions I have with my lab mentor, the Ph.D. students I am working with, and the students in my program who are becoming invaluable friends.

Week 2: University of Chicago Medical Center

June 22nd, 2012

Jordan Kemme ’13, Becker Fellow in Neuroscience

It is now my 10th day working at the University of Chicago Medical Center. Arriving last Monday felt reminiscent of New Student Orientation at Cornell as I walked into Max Palevsky Resident Hall to be greeted by peppy RA’s, medical students that help us through the transition here. After I checked in I began the work intensive process of moving my things from my car to my room, a suite that I share with two other guys. The next two days were reserved for orientation purposes. We covered basics that ranged anywhere from patient privacy laws to the benefits and dangers of living in Hyde Park. I haven’t encountered any sketchy situations running around the city yet and, in fact, running along Lakeshore Trail offers a wonderful breeze and Lake Michigan is a much needed change in scenery from Iowa’s endless cornfields.

My Cornell Fellowship was merged with an undergraduate program for summer research which has allowed me to meet many other youngsters like myself. The two day orientation was a great way to explore the campus and learn more about the University of Chicago and the Ph.D./M.D. programs. However, I am not here to follow doctors around the hospital every day; I am here to participate in scientific research and experience all the difficulties and excitement it can bring. After the orientation process was complete I was handed over to Dr. Richard Kraig (I’ll call him Rich), who’s laboratory focus at the moment is spreading depression, a slow spreading, self-propagating wave of electrical hyperactivity in the brain, and its role in migraine headache and the visual scotoma that sometimes accompanies it. I have only spent five days working in Rich’s lab and I have already begun to enjoy it. The first few days were comprised of reading numerous papers and chapters from a neuroscience text book so that, as Rich says, I will have the “Christie-Pope Neurobiology course before taking the Christie-Pope Neurobiology course”.

When I’m not chasing Rich around the hospital, vigorously attempting to keep up with his Olympic Race-Walking pace, I am at the desk reading papers. I have recently been aloud to perform some procedures on my own which allows me to show up before Rich in the morning while it is still quiet and peaceful. Rich has given me the task of exploring the role of microglial cells in the phenomenon of spreading depression. Microglia are supporting cells found in the central nervous system that perform macrophage-like activity in the presence of tissue lesioning or damage that likely initiate spreading depression. I have been working alongside a Ph.D. student, Aya Pusic, who, along with Rich, has been teaching me the laboratory procedures and the fundamental ideas behind our work. Everyday I learn more about the different cells that compose the nervous system as well as the broader image they all form to create the nervous system and the numerous, complicated functions they carry out. In the future I am looking forward to completing the steps necessary to perform my experiment that will hopefully shed more light on migraine headache, an ailment that affects 30 million Americans. For now, I’m content with the lessons I receive from Rich who seems to uphold the same ideals of “tough love” as several of the biology faculty back at Cornell.

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