The hypothesis for this proposal is that increased age results in increased susceptibility of cartilage to traumatic injury, which can be immediately imaged in vivo using multiphoton microscopy (MPM). For these studies, traumatically injured cartilage from horses of different ages will be optically assessed for chondrocyte and matrix damage in live tissue. First, damage resulting from injury will be mapped using MPM in an ex vivo model in order to minimize experimental variability; this information will then be validated in vivo using MPM to detect early damage. If successful, this will be the first study to use a non-destructive live imaging technique to define chondrocyte damage at the cellular level immediately following impact injury. This information will not only enhance our understanding of the pathogenesis of post-traumatic osteoarthritis (PTOA), but it will aid in the development of therapeutic strategies to prevent the development of OA.

Aim 1. Evaluate the effect of age on chondrocyte changes in early PTOA using MPM and transmission electron microscopy (TEM). Aging is the most significant risk factor associated with OA. Osteochondral (OC) blocks from young and adult horses will be subjected to traumatic injury and imaged immediately with MPM to quantify the development of autofluorescent optical signatures that our group has identified as related to cartilage damage (Appendix 1). The difference between intracellular organelle morphology after injury in young and adult animals will be compared using TEM, particularly from those areas densely populated with autofluorescent optical signatures.

Aim 2. To validate an in vivo large animal PTOA model that will enhance our understanding of PTOA in racehorses. Traumatic loading will be delivered to the cartilage surface in vivo and evaluated using arthroscopically-adapted MPM. Immediately after injury, damaged cartilage will be imaged using MPM and TEM as described in Aim 1. Replication of ex vivo methods to an in vivo scenario is not always direct, so there is a need to validate ex vivo findings in the in vivo situation. Use of an in vivo model is necessary to define long-term effects of impact injury and to evaluate potential strategies to prevent the development of PTOA.    

     The information gained from the studies in this proposal is important for understanding the very early initiating events of PTOA and for the development of approaches − medications, or alterations in exercise protocols − that are aimed at preventing the development of OA.