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In my internship I will be assisting Dr. Stephen Schlecht and his team in their research on the anterior cruciate ligament. The main objective of the research I worked on was to determine the mechanism that contributed to the bone loss in mice that underwent an anterior cruciate ligament rupture (ACL). The three mechanisms that were explored are tested by SHG, IHC, and histology to discover the most plausible mechanism. By the completion of this research it is expected that the results will be applicable to contribute to the understanding of the high rates of ACL graft failure in humans, specifically adolescent females.
Project Summary
Project Summary
The anterior cruciate ligament (ACL) stabilizes the knee and resists forward displacement of the tibia relative to the femur. ACL injury rates in the young are increasing, with ~50% of patients developing knee osteoarthritis within 10 years. Moreover, young patients are six times more likely to suffer an ACL graft failure within the first five years of reconstructive surgery compared to adults. Using an in vivo ACL rupture animal model, we investigated the physiological response to injury within the bone, ligament, and synovium of the knee. Previously, the Schlecht Lab showed significant bone loss within the ACL attachment in young adult female patients. Based on this outcome, we hypothesized that bone loss would be greater at the enthesis compared to the whole joint due to localized disuse osteoporosis. To test this, we ruptured the ACLs of adolescent C57BL/6J inbred mice in vivo, followed by euthanasia across multiple post-rupture time points. The loaded and non-loaded knee joints were then three-dimensionally imaged with micro-computed tomography (µCT) and processed for immunohistochemistry. My role in this study has been to 1) validate µCT region-of-interest selection, 2) perform general staining and immunohistochemistry, 3) image and quantify antibody presence in tissue sections, and 4) conduct gait analyses and quantify video-captured gait pattern fluctuations in mice across 28 days post-rupture.
Elevator Pitch
Elevator Pitch
Contributions
Contributions
Bone Analysis
Bone Analysis
One of the first projects I worked on was computing the area of the two types of bone. This is an important factor in the research since it willbe able to show if the rest time between the days of testing impacted the amount of bone damage in the mice. This is comparable to the rest days that athletes undergo during training periods.
Bare ACL Slides
Bare ACL Slides
The ACLs and surround portion of the knees including the femoral and tibial condyls are disected from the mice and then tested with various agents to analyze wether particular proteins are present and to what amount along with the number of alive and dead cells.
IHC Tolene Blue Slide
IHC Tolene Blue Slide
At the beginning of my internship my main responsibility was taking microscopic images of the ACL slides stained though IHC methodology with toluene blue. This process distinguished the enthesis from the bone and the ligaments as well as counterstaining that showed individual cells based on the properties of the different tissue.
Gait Analysis
Gait Analysis
Gait analysis was done on 15 mice pre-rupture and post rupture on days 3,7,14, and 28.
Differences were found in toe spread between the ruptured and unruptured knee. Although this finding was significant, it was determined that loading was done on both knees, therefore immobilization of the ruptured knee was not a causality for bone loss
TUNEL- DNA Processed Image
TUNEL- DNA Processed Image
TUNEL imaging is used to signal apoptosis. The region of interest is the enthesis in the cortical bone of the femur. But, no significant change was present between alive and dead cells in the control and rupture ROI.
Poster
Poster
Learning and Skills
Learning and Skills
Through this experience I will learn about the technicalities regarding the research process. I will also develop skills in using a microscope for histology purposes and analyzing the images. As this experience continues, I will have more responsibilities and will learn how to use different tools in the lab.
Histology Imaging
IHC Staining
Bone imagining computer program knowledge
Humane animal (mice) technique in lab
As the semester is coming to a close, my skills and responsibilities in the lab have greatly increased. I have learned more about the inner workings of what pre-work goes into running an experiment. It has been very eye opening to see what Dr. Schlecht, his team, and I have been working on come to life.
How did your contributions impact the larger project or goals at your site? Why did your work matter to getting the bigger job done?
How did your contributions impact the larger project or goals at your site? Why did your work matter to getting the bigger job done?
My contributions have greatly impacted the overall progress of the project as I have been filling multiple roles and contributing to the research by completing various tasks. Initially, my main focus was imaging the toluene blue stained slides for analysis of the enthesis regions on the femur and tibia. Next, I validated the regions of interest on the femur and tibia through micro CT. After this, I did various stainings of slides for the observation of antibodies. Once this was completed, I manually counted the presence of dead and alive cells in the areas of interest. This was crucial to the study because these findings led to the discovery of the mechanism that may contribute to bone loss following an ACL tear. My last role was measuring and analyzing gait patterns in the mice at various points post rupture. This data was necessary to rule out that the presence of a change or absence of change in gait pattern contributes to the change in bone loss.
Where else would you like to grow? What actions will you need to take in this role, or what other experiences after LHSI will you need to seek out to strengthen those skills?
Where else would you like to grow? What actions will you need to take in this role, or what other experiences after LHSI will you need to seek out to strengthen those skills?
From the spring self-evaluation, I have grown in my independence and reliability. I am able to prioritize, plan, and meet my requirements in the time frame set by my supervisor. Since I typically work independently on all of my projects, I have grown in my ability to adapt and apply previously learned techniques to a variety of situations. When I face a challenge, generally I am able to solve it on my own, but occasionally I have to reach out to receive assistance. Since my work has consisted of independent tasks, I would still like to develop my interpersonal skills in collaborating with others.
The Workplace
The Workplace
How would you describe the workplace culture at your internship site using some of your responses to the questions in the article?
How would you describe the workplace culture at your internship site using some of your responses to the questions in the article?
The culture at my internship site is very independent. There are only four of us at this site and we all have our own individual roles. It has been interesting to see how each of our projects have converged throughout the semester and the formation of the research, especially with the writing of the paper. Even though the day to day activities in the lab are independent, I would still describe my workplace culture as collaborative.
In the workplace I image slides of mice femurs and tibias to compare the cartilage, bone, tendons, and ligaments. I have also organized data in excel, stained slides, and done micro CT analysis.
How would you describe your ideal workplace culture? Discuss any differences between the workplace culture at your internship and your ideal workplace culture.
How would you describe your ideal workplace culture? Discuss any differences between the workplace culture at your internship and your ideal workplace culture.
I feel the environment at my current internship site does not necessarily exhibit my ideal workplace. Although I like independent work, I enjoy the few days that we have brainstormed together and collaborated to solve issues. These days have been few, resultantly, I have realized how I value a worksite where group work is the main focus.
After working here for two months, I have learned how important communication is as a professional. At my internship site many different studies are goin on at once and everyone's information is important to each study. A skill that I would like to continue to work on is using my professional communication to collaborate efficiently.
When entering my internship I was under the impression that I would be working on one thing at a time and have a specific project to work on. Contrary to my belief, in the first semester of my internship I had many different tasks which contributed to the study in different ways. I also did not realize how many different steps there are in the reseach process and how sometimes you have to do a multitude of processes before you have the final product that is going under anaylsis in the research.
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