January 27th, 2014

Today, I observed perfusions for the first time. Although I have experience dissecting animals in previous classes, I have never been a part of the animal’s death. I was very interested in learning about the process in general. In regard to the perfusions, I struggled most with how I was supposed to respond to the process. On one hand, I was curious. On the other, it was an eery experience to watch the animal die in front of your eyes.

The process was easier for me to digest when I thought of the rat as a research specimen. Once I was able to compartmentalize my thoughts, I thought the entire procedure was fascinating. Particularly when Dr. Holstein injected the rat with formalin, it was very interesting to watch as the rat’s muscles seized. It was also interesting to watch as Dr. Holstein extracted the brain from the rat’s skull. It was a lot less gruesome than I imagined because the blood was essentially drained from the rat and replaced with saline and eventually formalin. 

I also helped Bekah clean out the machines and sweep/mop the floors of the testing room. Tomorrow, we plan to go over the results and discuss our presentation. 

January 26th

Today, I came in and gave two rats water for 15 minutes.

January 23rd, 2014

Today I began the day by setting up the computer. The first round of injections (D1- all receiving quinine) went smoothly for the aCSF. However, we needed to redo the left injection for the first CDP rat. After Bekah and I flipped the water for group C, we agitated the cannulae for the rats in A and B groups. Dr. Holstein was concerned that the cannulae could be clogged, which could be problematic when they inject the dye for the perfusions. The second round of injections also went well for D2 (all receiving quinine). Similar to the first set of injections, we needed to redo the left cannulae for the CDP rats. We also labeled the bottles for the brains. D3 also went smoothly. However, we had to redo the left cannula for the rat in box two and three.

January 22nd, 2014

Today, I started off the day with C1-receiving all quinine. The aCSF injections and the first CDP injection went smoothly. However, we had to redo the right injection for the second CDP rat. We noticed that the second and the third box had very low licks. The second round of injections (C2-all quinine) also went smoothly, especially after we let the CDP run and scraped the edges of the injector before inserting them into the cannulae. I also made up 4 liters of NaCl and made 17 syringes of ketamine.

January 21st, 2014

Today, I began the day by setting up the computers for the day. The first set of microinjections (D2-all quinine) went smoothly. We only had to redo one injection. More specifically, we had to redo the left cannula of the second CDP rat. After D2, we flipped the water for group C for an hour. In the second set of microinjections (D3-all quinine), the aCSF microinjections went off without a hitch. I noticed that these rats were very fidgety and we had a difficult time keeping them still. Both CDP injections had to be redone. It seems that there is a problem with the tubing! After the D3 microinjections, I made up a liter of NaCl for the afternoon and helped Bekah make up extra syringes of LiCl for the CTA testing.

January 20th, 2014

Today, I began the day by making up .03 NaCL solution. We got off to a slow start in the first round of microinjections on C2, where the rats received quinine. Although the aCSF microinjections went smoothly, the CDP injections proved to be more problematic. Afterward, Bekah and I agitated the cannulae and weighed the rats in A and B. The aCSF microinjections also went smoothly for the rats in C3. However, we had to redo the microinjections for the CDP rats. The rats in boxes 1-3 received quinine, whereas the rat in box 4 received NaCl. We also prepared the LiCl solutions for the CTA tests. Dr. Holstein also explained the perfusion process and what to expect. The microinjections for the C3 group, receiving quinine, were successful and we did not need do redo any of them!

January 18th, 2014

Today, I came in to give water to rats in C and D for an hour.

January 17th, 2014

Today we will complete the microinjections for group D. The rats in D3 received .05 and .3 NaCl. The aCSF microinjections went smoothly. However, both CDP rats had to be redone (one in both cannulae, the other in just the left cannula). Bekah and I flipped the water bottles for the rats in group C for an hour. We experienced some difficulties during the second round of microinjections (D1), where the rats received received saccharin. The tubing was not cooperating for the acsf or the cdp rats. Bekah and I agitated the cannulae for the rats in A and B. Hopefully, the third set of microinjections will go smoothly for the afternoon group. Tomorrow, I will come in and flip the water bottles for the rats for an hour.

January 16th, 2014

Today we completed microinjections for group C (1, 2, 3). The first set of microinjections (C3) went smoothly; each rat received .01m saccharin. Unfortunately, we had to make a note that C3 was ran under the label C1. After C3, Bekah and I flipped the water for fifteen minutes for group D. The rats in C2 received .1m MSG. Rat 30 had a good left microinjection and we needed to redo the right microinjection. We also noticed that there was a little blood coming out of the left cannula following the microinjection. Later this afternoon, someone will have to agitate his cannulae. Following the C2 microinjections, I weighed out Saccharin, MSG and NaCL solutions for tomorrow. We finished up the morning microinjections with C1. In this group, boxes 1-3 received MSG and box 4 received saccharin. Bekah and I also agitated the cannulae for rats in group D.

January 15th, 2014

This morning we completed microinjections for three groups of rats (Group D: 1, 2, 3). I weighed out solutions of MSG and Saccharin. The first set of microinjections (D1) went smoothly; all rats received saccharin. Fortunately, we didn't need to redo any of them! Following the first set, Bekah and I flipped the water for all rats in group C for one hour.The second and third set also went smoothly. All rats received MSG in D2 and all rats in D3 received saccharin. After the third set of microinjections, Bekah and I replaced the cages for rats in group C. We noticed that one rat had the L and R cannula switched, so we had to make note that there had been a mix up at some point. Also, we gave all of the rats in D fifteen minutes of water. I'm looking forward to tomorrow!

January 14th, 2014

This morning we completed microinjections for three groups of rats (Group C: 1, 2, 3). The schedule is much more complicated, as we have added MSG and saccharin solutions. As a result, we all had to be much more vigilant when checking the solutions during the microinjections. We also needed to make sure that we double checked the amount of fluid required for the solution, as some of them need 400ml of DI water as opposed to 200ml. Group C's injections went smoothly. However, we needed to redo one of the injections for two rats. It seems that the cannulae are getting stopped up. After the microinjections, Bekah and I agitated the cannulae for the rats in group D. Two of the rats in group D have clogged cannulae and Sarah had to rinse the cannulae with ethanol. Tomorrow will definitely be a busy day!

January 13th, 2014

Today was a pretty easy day. I went in and agitated the cannulae for all of the rats and I made "do not water" tags for all of the cages. Tomorrow, we will have a very busy day and will complete microinjections for three groups of rats in the morning.

January 12th, 2014

Today, we worked with the rats in B1 and B2. The microinjections went relatively smoothly. However, it took a while to prepare the tubing for the CDP rat in B2. I also learned how to vortex the CDP and aCSF. After watching Dr. Holstein and Alex, I completed my first microinjection on Rat 10. This rat has a damaged left cannula but he will be undergoing microinjections each day. Dr. Holstein and Alex taught me how to hold the rat during the microinjection. Because I have small hands, Alex held the rat's bottom during the injections. I have to say, it was a very long four minutes. Even though I have gotten very comfortable inserting and removing the dummies in the cannulae, I found it much more difficult during the microinjections. The rat kept writhing around and I had to consciously remind myself to keep the tubing away from the rat's mouth. After we completed the microinjections for the first set of rats, I cleaned the tables in the lab and swept the floors. I also agitated the cannulae for the rats, excluding those who underwent surgery today. At the end of the microinjections, Alex and I cleaned out the water bottles.

January 10th, 2014

Today I began my day by mixing up cement for the surgeries. I prepared syringes of ketamine and atropine. I also weighed the rats who had not undergone surgery. A majority of the rats are now over 300grams, so they will be ready for surgery soon. I also helped trim the dummies to the size of the cannulae. When I was preparing the cannulae, I used a sharpie to mark the bottom edge of the cannula. The marking will remind whoever is performing the surgery not to get cement above the marking. On Sunday, I will work with Dr. Holstein and Alex on the microinjections.

January 9th, 2014

I began my day by measuring out sucrose and citric acid solutions for the rest of the week. I also set out DI water and the waste beaker for the microinjections. Dr. Holstein also taught me to set up the computers for the microinjections. On the computer, I entered the weight of the water bottles and the assigned concentration and solution for each rat. Afterward, I weighed the rats (without cannulae), who are steadily gaining weight. I was able to watch Dr. Pittman perform surgeries on two of the rats. Also, I watched as Alex prepared the Ketamine syringes for sedation. Alex also taught me how to prepare cement for the surgeries. Tomorrow morning, I am going to help Dr. Pittman by preparing cement for the surgeries.

January 8th, 2014

January 8th, 2014
Today I worked on handling the rats and I am much more comfortable moving the dummies in and out of the cannulae. I also helped label the rat boxes based on the rat's assigned group (A1, A2, B1, and B2). I also helped to mark the rats' tails. Also, Reid and I weighed the rats without cannulae. I also learned about microinjections today. I know that it is going to be a tedious process because so many factors can influence the success of the injection. I was surprised that even Dr. Sarah had some difficulties avoiding air bubbles in the tubing. Tomorrow, I am going to work on mixing solutions for the first phase of microinjections.

January 7th, 2014
   Today marked the first day of my research interim. I began my day learning how to mix sucrose solutions. Alex showed me how to measure the sucrose on the scale, add it to the specified amount of DI water and mix it on the stir plate. Today’s solution had a concentration of.2 moles of sucrose. We added 68.46g of sucrose in one liter of water. We proceeded to divide the solution among the water bottles and attach rubber bands to ensure that the tops stayed in place.
   Afterward, we learned how to handle the rats and agitate their cannuli. Although there are multiple ways to handle the rats, I preferred to use my index finger and thumb to hold the rat still. Inserting the dummy into the cannula proved to be a challenging task for multiple reasons. For one, the hole of the cannula is so small! Additionally, I was worried about bending the dummy, especially as the rat writhed around. I am excited to report that I was able to remove and replace the dummy on two rats! We weighed all of the rats, including those who had not undergone surgery. Based on their weight, Dr. Pittman selected the heaviest rats without cannulas. Today, he performed surgeries on the rats and I was able to watch!
   After we handled the rats, we went into the dark room and weighed the eight water bottles. We accidentally recorded water bottle number 4 as number 1, so we had to make a special note for future reference. We began testing and returned after an hour had elapsed to weigh the water bottles again.