In Vivo Fluorescence Reflectance Imaging to Quantify Sex-Based Differences in Protease, MMP, and Cathepsin K Activity in a Mouse Model of Post-Traumatic Osteoarthritis.

摘要

Post-traumatic osteoarthritis (PTOA) is a frequent result of traumatic injury to the joint, which largely includes partial and whole tears to the anterior cruciate ligament (ACL). Over 50% of individuals that experience ACL rupture have developed PTOA within 10-20 years, resulting in severe joint pain and stiffness. Although females are 4-6 times more likely than males to sustain an ACL injury during increased activity, males demonstrate an increased tendency to develop osteoarthritis following injury. ACL rupture is characterized by a surge of inflammatory cytokines, matrix metalloproteinases, and other proteases, as well as cartilage degeneration and rapid bone turnover. Preventing these inflammatory processes may be vital in suppressing the prolonged development of PTOA. In this study, we attempt to quantify the difference between male and female mice development of PTOA resulting from a non-invasive injury model, in which a single load of tibial compression overload is performed to rupture the ACL.;We utilize this model to determine the time course of protease activity, inflammation, and osteoclast resorption activity in osteoarthritis and how they differ between males and females by using highly sensitive activatable fluorescent agents.;3 groups of 16 mice (8 male, 8 female) were injured via tibial compression overload, and each group was injected with ProSense 680, MMPSense 680, or CatK 680. Injections were administered 24-30 hours prior to imaging, on days 1, 3, 7, 14, 21, 28, and 56 after initial injury. To quantify fluorescence levels, imaging was performed with the IVIS Spectrum system. Levels of protease activity, inflammation, and osteoclast activity were measured by ProSense 680, MMPSense 680, and CatK 680, respectively, by evaluating radiant efficiency of the signal within a uniform region of interest (ROI), anatomically selected around the knee on grayscale image. Radiant efficiencies of the injured knees were normalized over the contralateral uninjured knees and compared through the entire time course to assess the variation of activity.;For both male and female mice, protease activity (ProSense), MMP activity (MMPSense), and osteoclastic bone resorption (CatK) were significantly increased in the injured knee relative to the uninjured knee at nearly all time points. For example, protease activity was significantly increased by day 1, reached a peak between 3 and 14 days, and then decreased at later time points (Figure). Males and females displayed similar changes in injury response in looking at protease activity and MMP activity through the time periods observed, with males averaging a slightly higher normalized radiant efficiency at early time points, although this was not statistically different. Osteoclastic bone resorption activity demonstrated no discernible trend between males and females.;Using commercially available activatable fluorescent agents, we were able to quantify the time course of protease activity, MMP activity, and osteoclastic bone resorption in male and female mice following traumatic knee injury. However, contrary to our hypothesis, we were not able to observe a significant differential response between male and female mice. Our future studies will continue to explore potential mechanisms of the sex-based adaptation to joint injury that may contribute to the greater incidence of PTOA development in males. Our future studies will also continue to use fluorescence reflectance imaging as a method for measuring biological activity in vivo.