ADVANCED TISSUE CLEARING APPROACHES FOR 3D PATHOLOGICAL BRAIN INVESTIGATION

Characterizing the three-dimensional (3D) cyto- and myeloarchitecture of the human brain remains a major challenge due to its complex cellular organization and dense connectivity. Recent advances in 3D fluorescence microscopy, combined with optical tissue clearing and high-performance image analysis, now enable volumetric visualization of intact neural tissue at unprecedented resolution and scale. Here, we present multiple approaches based on optimized tissue clearing and multiplexed fluorescent labeling, which, combined with advanced microscopy, enable the generation of comprehensive 3D maps of neuronal organization and fiber architecture in both human brain specimens and whole animal brains. We optimized iDISCO, SHORT, and tissue expansion clearing protocols (SHORT-EX) for compatibility with multi-target immuno- and FISH staining and applied fluorescence microscopy for multi-resolution volumetric imaging. These approaches enabled detailed reconstruction of neuronal spatial distribution, cellular morphology, and fiber orientation, allowing a deeper understanding of brain anatomy. We analyzed both healthy and pathological human brain samples, including Focal Cortical Dysplasia and Alzheimer’s disease, as well as whole mouse brains from Huntington’s disease models, and honeybee brains to assess the neurotoxic effects of microplastics. Quantitative 3D image analysis revealed disease-specific alterations in cytoarchitecture, molecular pattern distribution, and tissue organization. Overall, our results provide valuable anatomical insights into neural organization and pathological remodeling, offering a powerful framework for studying structural brain alterations in health and disease.