3D Light Sheet Microscopy Reveals Aging Osteoarthritis-associated Joint-innervated Nerve Remodeling in Mouse Knee Joints

Abstract Objectives Osteoarthritis (OA) is a prevalent age related joint disease-causing chronic pain. This study investigated how nociceptive and sympathetic nerve innervation in the mouse knee joint changes with age and OA progression, and how these changes relate to pain and disease severity. Methods Thirty-eight mice were assigned to four groups: young male (M y ), aged male (M a ), young female (F y ), and aged female (F a ). Pain sensitivity was evaluated via Pressure Application Measurement (PAM), and joint damage was graded using OARSI scoring. CGRP and PIEZO2 expressions in dorsal root ganglia (DRG) were also assessed. We employed iDISCO tissue clearing and 3D light sheet fluorescence microscopy to visualize total (PGP9.5⁺), nociceptive (CGRP⁺), and sympathetic (TH⁺) nerve fibers in anterior regions of mouse knee joints. A MATLAB-based tool quantified nerve architecture. Results The M a group displayed the highest OA severity and markedly lower mechanical withdrawal thresholds compared with M y (H = 11.59, ε² = 0.64), suggesting an age effect on pain-related behavior. This phenotype was accompanied by a higher total PGP9.5⁺ nerve fiber density in the knee joint (mean difference −0.3127, 95% CI −0.4551 to −0.1704) and increased CGRP⁺ nociceptive innervation. In contrast, female mice showed no age-dependent change in PAM withdrawal thresholds, consistent with preserved cartilage integrity and stable OARSI scores, and no detectable age-related differences in PGP9.5⁺ or CGRP⁺ innervation. The TH⁺ sympathetic fiber distribution was comparable across sexes and ages. Consistent with joint-level findings, DRG analyses demonstrated increased CGRP and PIEZO2 expression in the M a group, whereas females exhibited no significant change. Conclusion Enhanced nociceptive but not sympathetic nerve remodeling in knee cavities is associated with increased OA severity and knee pain in the M a group. These findings emphasize the role of peripheral sensory plasticity in OA pain and demonstrate the value of 3D imaging for visualizing neuroanatomical changes in joint disorders.