A Novel Rapid 3D Tissue-Clearing and Staining Approach for Enteric Neurovascular Imaging and Pathology Applications

Background and Aims: Neurovascular abnormalities, such as aberrant nerve migration in Hirschsprung’s disease and reduced vascular density in necrotizing enterocolitis, are frequently observed in intestinal diseases. Traditional 2-dimensional (2D) staining methods are complicated, time-consuming and fail to comprehensively visualize the intricate neurovascular structures and morphology of the intestine. This study focuses on evaluating a novel 3D staining technique that promises simpler, faster, and more effective visualization of intact neurovascular structures in the colon. Additionally, it aims to compare the strengths and limitations of this 3D method against traditional 2D techniques for analyzing neuronal and vascular changes in two prevalent pathological conditions. Methods: A novel tissue-clearing approach was used to render mouse and patient distal colon tissues transparent. Neural structures and blood vessels were stained. 2D and 3D imaging were performed with laser confocal or tiling light sheet microscopy. Parameters include total imaging time, imaging range, image quality, operational complexity, and post-processing were compared between 2D and 3D methods. Results: Compared to 2D imaging, 3D imaging reveals the complete morphology and trajectory of neurovascular structures. Confocal 3D imaging offers superior clarity, higher transparency, and faster workflow efficiency, whereas light-sheet microscopy provides broader coverage at the expense of lower image quality. Post-processing facilitated spatial modeling and quantitative analyses. Applications included Hirschsprung’s disease, where 3D imaging revealed abnormal nerve distribution, and congenital heart disease, where hypoperfusion impacted vascular development in the colon. Conclusions: Confocal 3D staining and imaging offered a more streamlined workflow and enabled comprehensive visualization of neurovascular architecture, supporting efficient assessment of intestinal neurovascular phenotypic features.