Keeley Garvey, Week 1, von Frey X 320

View from my desk

My first week at Duke has been exciting and informative. On the first day, I entered the Snyderman Genome Sciences Research Building 1 with chocolate and nerves. My grandmother is Italian and insisted that I come with gifts on the first day. The grad student I am working with, Mr. Yul Huh, told me not to be nervous and gave me a tour of the lab. It was all very overwhelming at first, but he made me feel comfortable. He told me that a student from Dartmouth started a week earlier than me and would be working with me in the lab. After lunch that day, Shawn, the student from Dartmouth, sent me a few primary articles that would be necessary to read because they are the foundation of my project. For the most part, Shawn and Yul are the ones I go to if I need help or don't understand something because there is somewhat of a language barrier in my lab. However, there are a few other labs next to us that are doing similar pain research, so I could go to them as well. For the rest of the day, I read the articles because Yul had to edit his paper for a grant.

GSRB1

On Tuesday, we received 10 of our wild type CD1 female mice. We were supposed to get males as well, but there was a mess up somewhere along the line. After lunch, Yul showed Shawn and I how to do two types of behaviour testing on the mice. The first test, the frequency test, is a type of test where we use a certain type of filament (0.4) to test the sensitivity of the mice- this is part of a test called the von Frey test. In this behaviour experiment, each mouse is "poked" with a 0.4 filament on a certain area of its left hind limb. We are careful not to test their toes and heel because they are known to be extra sensitive. We "poke" each mouse 10 times with the same filament and mark each response down. An X represents a response, and an O represents no response. From the results, we were able to determine the frequency, or percentage, of X's from each mouse. As it turns out, the average frequency of sensitivity was about 30%. This is very likely to change after we treat the mice with a cancer drug called paclitaxel. Treating the mice with this drug is the foundation of our experiment because it is found that drugs that treat cancer elicit chronic pain in patients. Eventually, Shawn, Yul, and I will treat the mice with paclitaxel and determine if they are more sensitive or not. We expect to see that the frequency of sensitivity will increase dramatically because the mice should be more sensitive. The other test, called the up-down test, is somewhat similar to the frequency test. For this experiment, we test the mice with 5 different sizes of filaments (.16, .4, .6, 1.0, and 2.0)- .16 being the weakest, and 2.0 being the strongest. In this test, each mouse is "poked" with the weakest filament. If there is no response, we mark an O and move up to the next strongest filament. If there is a response, we mark an X and move back down to the weaker filament and test that again. We do this 6 times to determine the mouse's sensitivity. Each series (ex: OOXOO) corresponds with a number- I am not sure how this works yet. Shawn and I tested the mice 160 times that day. 

von Frey filaments

CD-1 wild type female mouse

Shawn testing the mice

On Wednesday, Shawn, Yul, and I "sacrificed" and dissected the mice. Because Shawn and I are not allowed to "sacrifice" the mice, Yul had to do so. 5 female mice were put to sleep using isoflurane (anesthesia) and their heads were chopped off using a guillotine. Yul first showed us how to dissect the mice properly so Shawn and I could correctly extract the bone marrow. First, we cut down their back so the their spine could be exposed, then we cut the skin down their legs. Next, we cut the hind legs off by cutting down the spine and stopping at the hip bone. Then, we would take the skin and hair off. After this, we cut off the foot at the ankle and split the leg in two sections- 1) femur, 2) tibia/fibula. Then, the excess muscle and connective tissue was cut away from the bones so they could be exposed. To make it a little easier, the tips and ends of the bones were cut away so the bone marrow could be easily accessible. We put the bones in PBS (buffer solution) so the bone marrow cells would stay alive. When all the bones were taken out and put in the buffer, we took the bones to the tissue culture room. This room has many "hoods", which are like metal-covered desks that keep whatever is on the desk sterile and free of contamination. Yul showed us how to do the following because it was late and he did not want it to take too long. First, media and antibiotics were put in petri dishes so the cells would have food and protection. Then, the bone marrow was washed out of the bones using media and an equal amount of solution was plated into the petri dishes. We incubated the cells overnight and hoped that the extra cells in the solution would be removed. On Thursday, we took out the media and antibiotics from the dishes and the bone marrow cells were not removed because they stuck to the plastic dishes. We removed the solution because each day, the cells need to be replenished. After new media and antibiotics were given, we reviewed the cells under the microscopes. As it turns out, our cultures have been growing exponentially and our work has not been in vain. Because our male mice have yet to arrive, we had to go into the -80 degree freezer and take out male bone marrow cells that were extracted before we came so they could be cultured. On Friday, we started testing 10 mice with the von Frey test and frequency test. Shawn, Yul, and I all tested the mice 160 times again and then compared our results at the end of the day. Surprisingly, me and Shawn's results were more similar than Yul's results which showed that the mice were more sensitive than usual.

When I was not in the lab:
During lunch breaks, Shawn, Deja, Yul, and I go out to a place called West Union on Duke's campus. The food there is pretty good and a lot better than other college food. Unfortunately, I do not have the budget to go out and eat each day, so I am going to start bringing a packed lunch to West Union so I can still be with my friends. On Friday during our lunch break, Shawn, Deja, and I all went to Shawn's hotel (Washington Duke Inn and Golf Club- aka the WaDuke) and Shawn and I went swimming. On Saturday, Deja and I went to the WaDuke to swim and hang out with Shawn until we all went to a trampoline park called Defy Gravity. After dinner, we went to an ice cream place called the Parlour in downtown Durham- the ice cream was really good. Today, we are going to the mall and I will be going out to eat with one of my best friends from middle school who lives in Raleigh. Overall, I have been loving my time in Durham- both in and out of the lab.