3D Light sheet microscopy reveals aging osteoarthritis-associated joint-innervated nerve remodeling in mouse knee...

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.

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.

Aged males exhibited the greatest OA severity and significantly lower PAM withdrawal thresholds compared with young males, indicating increased pain sensitivity with age. This phenotype was accompanied by marked remodeling of the total PGP9.5⁺ nerve network in the knee joint. Specifically, aged males showed approximately 2.0-fold greater total PGP9.5⁺ nerve fiber length and 3.6-fold higher PGP9.5⁺ branching complexity compared with young males. Nociceptive CGRP⁺ nerve fiber density was also elevated in aged males, whereas sympathetic TH⁺ fiber density remained unchanged across age and sex groups. Female mice exhibited no significant age-related differences in PAM withdrawal thresholds, cartilage degeneration (OARSI scores), or joint innervation. Consistent with joint-level findings, DRG analyses revealed ∼1.3-fold more CGRP⁺ neurons and ∼2.1-fold higher PIEZO2 expression in aged males compared with young males, while females showed no significant age-dependent changes.

Enhanced nociceptive but not sympathetic nerve remodeling in the anterior region of the knee 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.