{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,3,5]],"date-time":"2026-03-05T06:42:20Z","timestamp":1772692940447,"version":"3.50.1"},"reference-count":36,"publisher":"MDPI AG","issue":"5","license":[{"start":{"date-parts":[[2026,3,3]],"date-time":"2026-03-03T00:00:00Z","timestamp":1772496000000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"name":"National Children\u2019s Medical Center Young Clinical Scientist Program","award":["2022LCKXJ06"],"award-info":[{"award-number":["2022LCKXJ06"]}]},{"DOI":"10.13039\/100017551","name":"Children\u2019s National Medical Center","doi-asserted-by":"crossref","award":["EK1125180104"],"award-info":[{"award-number":["EK1125180104"]}],"id":[{"id":"10.13039\/100017551","id-type":"DOI","asserted-by":"crossref"}]},{"DOI":"10.13039\/100017551","name":"Children\u2019s National Medical Center","doi-asserted-by":"crossref","award":["EK112520180211"],"award-info":[{"award-number":["EK112520180211"]}],"id":[{"id":"10.13039\/100017551","id-type":"DOI","asserted-by":"crossref"}]},{"name":"National Clinical Key Specialty Construction Project","award":["10000015Z155080000004"],"award-info":[{"award-number":["10000015Z155080000004"]}]},{"DOI":"10.13039\/501100001809","name":"National Natural Science Foundation of China","doi-asserted-by":"publisher","award":["82305386"],"award-info":[{"award-number":["82305386"]}],"id":[{"id":"10.13039\/501100001809","id-type":"DOI","asserted-by":"publisher"}]},{"name":"Development Project of Shanghai Pek Disciplines-Integrated Chinese and Westem Medicine","award":["20180101"],"award-info":[{"award-number":["20180101"]}]},{"DOI":"10.13039\/100020732","name":"Innovative Research Team of High-Level Local Universities in Shanghai","doi-asserted-by":"crossref","id":[{"id":"10.13039\/100020732","id-type":"DOI","asserted-by":"crossref"}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["Diagnostics"],"abstract":"<jats:p>Background and Aims: Neurovascular abnormalities, such as aberrant nerve migration in Hirschsprung\u2019s 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\u2019s 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.<\/jats:p>","DOI":"10.3390\/diagnostics16050759","type":"journal-article","created":{"date-parts":[[2026,3,3]],"date-time":"2026-03-03T16:18:45Z","timestamp":1772554725000},"page":"759","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":0,"title":["A Novel Rapid 3D Tissue-Clearing and Staining Approach for Enteric Neurovascular Imaging and Pathology Applications"],"prefix":"10.3390","volume":"16","author":[{"ORCID":"https:\/\/orcid.org\/0000-0002-1495-0942","authenticated-orcid":false,"given":"Debao","family":"Li","sequence":"first","affiliation":[{"name":"National Health Commission (NHC) Key Laboratory of Neonatal Diseases, Department of Pediatric Surgery, Children\u2019s Hospital of Fudan University, Shanghai 201102, China"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Xuqing","family":"Cao","sequence":"additional","affiliation":[{"name":"National Health Commission (NHC) Key Laboratory of Neonatal Diseases, Department of Pediatric Surgery, Children\u2019s Hospital of Fudan University, Shanghai 201102, China"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Jienan","family":"Lin","sequence":"additional","affiliation":[{"name":"Department of Neonatal Surgery, The Affiliated Women and Children\u2019 s Hospital of Ningbo University, Ningbo 315012, China"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0009-0005-5531-3076","authenticated-orcid":false,"given":"Qingchi","family":"Zhang","sequence":"additional","affiliation":[{"name":"Xiamen Key Laboratory of Pediatric General Surgery Diseases, Children\u2019s Hospital of Fudan University (Xiamen Branch), Xiamen Children\u2019s Hospital, Xiamen 361006, China"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-9645-4768","authenticated-orcid":false,"given":"Rui","family":"Dong","sequence":"additional","affiliation":[{"name":"National Health Commission (NHC) Key Laboratory of Neonatal Diseases, Department of Pediatric Surgery, Children\u2019s Hospital of Fudan University, Shanghai 201102, China"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Song","family":"Sun","sequence":"additional","affiliation":[{"name":"National Health Commission (NHC) Key Laboratory of Neonatal Diseases, Department of Pediatric Surgery, Children\u2019s Hospital of Fudan University, Shanghai 201102, China"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Chun","family":"Shen","sequence":"additional","affiliation":[{"name":"National Health Commission (NHC) Key Laboratory of Neonatal Diseases, Department of Pediatric Surgery, Children\u2019s Hospital of Fudan University, Shanghai 201102, China"}],"role":[{"role":"author","vocabulary":"crossref"}]}],"member":"1968","published-online":{"date-parts":[[2026,3,3]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","first-page":"552","DOI":"10.1038\/s41583-018-0041-0","article-title":"Enteric nervous system development: What could possibly go wrong?","volume":"19","author":"Rao","year":"2018","journal-title":"Nat. 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