Project Summary Knee osteoarthritis (OA) is a painful chronic disease affecting 27 million people in the US. Knee OA is characterized by progressive damage and remodeling of all joint tissues. Major hallmarks of OA are joint pain and joint space narrowing on x-ray. Biomechanical and inflammatory factors play an important role in both joint pain and damage, but exactly how these pathways interact and promote mechanical sensitization of sensory neurons is unknown. Recently, we identified the sensory neuron mechanosensitive ion channel Piezo2 as a key contributor to mechanical sensitization in the destabilization of the medial meniscus (DMM) mouse model of OA. In addition, we have demonstrated that NGF-induced joint inflammation is dependent on nociceptor expression of Piezo2, suggesting that Piezo2 signaling may contribute to neuroimmune interactions. Finally, we have demonstrated that macrophage infiltration is a key contributor to the development of mechanical sensitization in OA. Nociceptor signaling helps to facilitate this recruitment of immune cells, but how the Piezo2 pathway is involved is not yet known. Therefore, this supplement represents an expansion of the existing Specific Aim 1 of the parent program. In particular, we will leverage our ongoing studies in nociceptor-specific Piezo2 conditional knock-out mice to now include examination of the neuroimmune changes happening in these mice, in both sexes. This will include using a novel imaging technology – light sheet imaging of cleared knee joint and spinal column/DRG tissue in order to visualize macrophages in these tissues in a volumetric representation. The research outlined here will fill the gap in knowledge by providing information on the interaction of inflammation and mechanical stimuli in producing mechanical sensitization. This proposal aims to answer the questions of: What is the role of Piezo2 in inflammatory mechanical sensitization? And, Can light sheet imaging be used to quantify macrophage numbers in the lumbar DRGs? We hypothesize that mechanical stimuli are necessary for inflammatory mechanical sensitization. In addition, we hypothesize that macrophage recruitment in the DRGs is a fundamental part of mechanical sensitization, and that recruitment will be inhibited by knocking out Piezo2. Successful completion of this supplement will improve our understanding of how nociceptors and immune cells communicate with the help of the Piezo2 pathway to promote mechanical sensitization in OA, which may lead to the identification of novel pathways to target both pain and joint damage therapeutically.