Project Summary/Abstract Chronic low back pain continues to exert a significant socio-economic burden on society as its contribution to the opioid crisis grows. This is because studies have failed to address the underlying mechanisms associated with pain generation. Intervertebral disc (IVD) degeneration is highly associated with low back pain (LBP), and neurovascular ingrowth into this usually avascular and aneural organ, has been identified in patients with chronic pain. However, the specific pathways propagating ingrowth and pain have yet to be identified. Mast cells regulate inflammation and pain in musculoskeletal diseases such as rheumatoid arthritis and osteoarthritis. Both our published and supportive work have demonstrated significantly increased levels of mast cell marker tryptase in the IVDs of human and canine patients with painful disc degeneration suggesting that mast cells function to enhance catabolism, inflammation and pain pathways in discogenic back pain. The overall goal of this proposal is to use clinically relevant and novel chondrodystrophic canine in vitro and in vivo animal models, to determine the role of mast cells and down-stream pathway protease-activated receptor 2 in LBP and how they can be modulated for therapeutic effect. This goal aligns well with high risk, high reward nature of the proposal as immune-modulation has never been applied to the IVD as a non-addictive strategy to treat LBP. Furthermore, the chondrodystrophic dog is currently under-utilized as an intermediate animal model of LBP despite its similarities to painful disc degeneration in humans and its relevance as a client-owned ?patient? population; this is largely because robust validation of behavioral assessments of pain are lacking. Our studies aim to address this gap while catalyzing innovation and improving scientific knowledge in this area. The first specific aim uses a novel high-through Bioreactor to investigate how co-culture of canine mast cell-nucleus pulposus under dynamic physiological load regulates catabolism, inflammation and neurovascular ingrowth in painful disc degeneration. In addition, mechanistic inhibition studies will be conducted to determine efficacy of FDA-approved drug Cromolyn sodium and protease-activated rector 2 antagonist on these mast cell induced effects. The second specific aim involves deep phenotyping the chondrodystrophic dog model of painful disc degeneration using a comprehensive assessment of pain behaviors (hyperalgesia, activity, posture and neuronal excitability) in addition to IVD structure/functional changes using histology, mechanics and MRI followed by screening with immune-modulatory drugs. These studies are both significant and innovative because they combine a unique multi-disciplinary team of medical and veterinary clinicians, a spine biologist, a neuroscientist, veterinary immunologist and histo-pathologist, and a biostatistician to investigate the role of the unexplored mast cell in the pathophysiology of LBP and in vitro and in vivo chondrodystrophic dog models for LBP. The broader impact of this proposal involves a pathway to clinical trials in client-owned dogs with LBP.