Comprehensive evaluation and application of tissue clearing techniques for 3D visualization of splenic neural and immune architecture

As the largest secondary lymphoid organ, the spleen plays a crucial role in initiating and sustaining immune responses against blood-borne pathogens through antigen capture and delivery. It is innervated by both autonomic and sensory nerves, which allows for neural modulation of its immune responses. The intricate spatial structure and precise coordination between immune and neural components is essential for proper splenic function, necessitating three-dimensional (3D) imaging to reveal its architecture. However, the dense fibrous capsule and exceptionally rich vasculature of the spleen pose significant challenges for achieving comprehensive 3D visualization of the entire organ. Here, we systematically evaluated and compared five cutting-edge tissue clearing approaches—ImmuView, FLASH, SHANEL, advanced CUBIC, and Ce3D—for their effectiveness in rendering the spleen transparent for multiplexed antibody staining and high-resolution 3D imaging. Our results indicated that SHANEL provided the clearest visualization of essential splenic neural and immune components. Meanwhile, advanced CUBIC achieved the greatest labeling efficacy for immune cells, albeit with slightly reduced transparency. Importantly, our study marked the first application of these optimized protocols to human spleen tissue, successfully revealing the highly organized immune cell zones and neural networks with enhanced clarity. Notably, we identified the nociceptive sensory innervation within human spleen tissue for the first time. Collectively, these findings establish optimal imaging strategies for visualizing splenic immune cells and neural structure in both murine and human tissues, providing profound insights into the intricate neuroimmune interactions and their pivotal roles in the immune functions of the spleen.