3D immunolabelling of human aortic valves by optimized CUBIC tissue clearing and its application for neural mapping in aortic stenosis

Calcific aortic valve stenosis (CAVS) is characterized by significant structural alterations in the aortic valve. However, its neuroanatomical landscape remains poorly understood. In this study, we developed an optimized three-dimensional (3D) imaging approach that combined enhanced Clear, Unobstructed Brain Imaging Cocktails and Computational (CUBIC) tissue clearing and SWITCH-Off/On to visualize the neuronal architecture within calcified human aortic valves derived from patients undergoing aortic valve replacement surgery. Standard CUBIC analysis protocols have shown limited efficacy owing to poor clearing and antibody penetration in mineralized tissues. By integrating SWITCH chemistry before decalcification and refractive index matching, we achieved uniform delipidation, fluorescence preservation, and deep tissue labelling. Light-sheet fluorescence microscopy enabled high-resolution 3D reconstructions with an 80% success rate, revealing heterogeneous and spatially organized neuronal distributions across the aortic valve. This method offers a robust platform for studying valve innervation and its potential role in mechanosensing and disease progression, opening perspectives for neuro-targeted interventions in valvular heart disease.