Evan Bradley, Post-Op Checks, Week 5

This week, most of the veterinarians and orthopaedic surgeons took a much-needed week off due to the hectic surgery days that occurred last week. I continued to work on my IVD degeneration disease load study with sections of canine cervical and lumbar spine. Furthermore, I monitored the eleven canine models that underwent full hip arthroplasties last week to make sure they were doing well during their post-op recovery period.

I received sections of canine cervical and lumbar spine from the veterinary medical diagnostic lab (VMDL) after a complete necropsy. This was my first time harvesting full sized canine IVDs out of the spine, which proved to be quite a different process than removing tail IVDs. Instead of using hemostats and bone-cutting forceps, I used a sterile bandsaw used specifically in the lab for removing full canine IVDs. The harvesting process included trimming the tissue and muscle that surrounded the spinal column in order to reveal where the IVDs were located. Then, I switched the standard bandsaw blade to a diamond-encrusted blade to cut out each individual IVD between the vertebrae. This blade grinds through the tissue rather than shredding it, which allows for a flatter cut that does not damage the fragile annulus fibrosus and nucleus pulposus. The blade requires constant irrigation, so I had to fill the saw base with a saline solution, which in turn kept the blade and tissue wet throughout the cut. Once the disks are removed, any excess tissue that was not completely removed during the initial cut is trimmed off and the IVDs are placed in beakers in a PBS solution. I trimmed the bone plates on either side of the IVDs with the smaller wet saw and set up them up in the Flexcell plates. Before enclosing the IVDs in the plates, I used a needle to inflict a simulated annular tear into half of the IVDs. This was performed by inserting the needle between the two vertebrae and using the syringe to pull some of the nucleus pulposus out of the annulus fibrosus. After, I tried to trim the bone plates as flat as possible on the wet saw so that the IVDs would stand up in the Flexcell plates. I was successful in doing so and was able to compress each IVD in the plate with the plastic inserts without any of them falling onto their sides. I then pipetted the DMEM/F12 media into the Flexcell plates through the plastic insert. I pipetted IL-1B into half of the wells to serve as the inflammatory cytokine treatment. This treatment will induce inflammation in the IVDs and will allow me to determine the effects of inflammation in the disks while they are under load. One plate was a .50 MPa loaded plate and the other was a no-load control. I placed the .50 MPa plate into the vice and attached it to the Flexcell vacuum system to begin applying a pulsating load. I developed a new regimen for the cervical and lumbar canine IVDs, which was very similar to the rat tail IVD "trucker" regimen, but with increased compression levels due to the significantly larger size of the canine spine IVDs. The no-load plate was placed alongside the loaded plate in the incubator for a 6-day culture. On day 3, I changed and collected the media for biomarker analysis at the end of the study. On the final day of culture, I performed the end-of-culture procedure, which included cell viability and viable cell density evaluations. I have performed this end-of-culture procedure many times and have developed and mastered new techniques for it, which have been added to the protocol to serve as a guide for the other undergraduate and graduate students. I am thrilled that utilizing the fluorescent microscope and cell counting program has become a part of my daily routine in the lab! I also measured each of the IVD's diameter with a caliper in order to determine the exact load applied to them during the culture. Dr. Stoker and I determined, through my previous Rat and canine IVD load cultures, that depending on the diameter of an IVD, the load applied to the Flexcell well varies. An IVD with a larger diameter receives a smaller compressive load compared to a smaller IVD due to it's increased ability to disperse the force applied to it. I mentioned this possible issue to him when I observed drastic viability differences between two IVDs of different diameters in identical treatment groups. He found an equation in one of the Institute's papers that accounts for this variability in compression based on disk diameter. This was the most complex IVD load model that I have completed so far due to the three different treatment groups involved. However, my results were consistent with my previous canine tail IVD load culture. The injured disks, exposed to IL-1B, and under the .50 MPa load showed the lowest cell viability and viable cell density. This was exactly what Dr. Stoker was looking for and he was especially ecstatic about the negative IL-1B effects on cell viability that I observed in this load model. Due to my results, IL-1B will now be used as the main inflammatory cytokine treatment in the lab and will be used on many different types of tissues in the future to determine its effects on non-IVD samples. Dr. Stoker is hopeful that the lab will be able to determine the link between these types of inflammatory cytokines and the development of osteoarthritis in the knee joint.

After the eleven full hip arthroplasties that our canine models went through last week, each of them had to be monitored on a daily basis in order to ensure they were not in any pain and their incisions were healing properly. AM and PM checks were assigned to the graduate students and I by Dr. Bozynski, the head veterinarian. I was assigned to do AM checks due to my inability to commute to the Animal Research Center (ARC) at night. The AM checks involved a temperature reading, heart and respiration rate measurement, and an incision site evaluation. Surprisingly, all of the dogs were standing and walking one-day post-op and all of them seemed to be in good health. The temperature and heart/breathing rate took less than five minutes to perform, and the evaluation was completed after a bandage change. As a result, the AM checks took me only 5 minutes to complete. All of the dogs' incisions seemed to be healing properly and there were no signs of infection around the staples. I notified Dr. Bozynski after each of my 5 AM checks and she collected my measurements for her records. She removed the staples with me after the 7-day mark. During the PM checks, the graduate students made sure to play and comfort each of the dogs to ensure they behaved themselves in their rooms overnight. Overall, the canines are doing very well and will be able to live a much more pain-free life with their new hip prosthetics.

I am still sitting in on all of the graduate student classes that Dr. Stoker teaches and attend a weekly journal club with Dr. Cook. We are continuing to learn about western blots and how they may be used in our lab for degradative enzyme identification. We have mainly focused on trying to identify aggrecanases in tissue samples from patients with severe OA of the articular cartilage from the femoral condyle and tibial plateau. I look forward to checking up on the canines next week and making sure they stay healthy and pain-free throughout their recovery process. I will continue working on the next rat tail IVD load model when the veterinary school delivers rat models next week.