Dynamic Pathology of Enteric Neural Network using Curcumin-assisted Multiphoton Laser Imaging in Hirschsprung Disease

Objective: In living tissue, it has been difficult to make microscopic-level observations without damaging the tissue.

Summary background data: We have invented a novel intravital fluorescent observation method (IFOM) for real-time tissue observation, combining multi-photon laser scanning microscopy (MPLSM) with curcumin vital staining (CVS-IFOM). The aim of this study was to use CVS-IFOM to analyze the enteric nervous system (ENS) in mice and human patients with hypoganglionosis and Hirschsprung disease.

Methods: In an initial viability study, we compared live ENS images from non-fluorescent C57BL6 mice stained with curcumin (n=5) and GFP mice (n=5) using MPLSM. We then explored CVS-IFOM for the live examination of resected colon tissues from one hypoganglionosis and three Hirschsprung disease patients.

Results: In the viability study, detailed ENS histological features were only observed in the curcumin-stained mice. In the hypoganglionosis patient, CVS-IFOM provided ENS details that were not visualized under H&E staining or calretinin immunohistochemistry, allowing the analysis of ENS size, neural bundle number, and neural cell number per plexus. In Hirschsprung disease patients, CVS-IFOM showed a gradual hypoplastic change in the ENS from the oral wedge to the anal wedge, detecting disproportionate changes in the ENS within the same intestinal level, supporting a circumferentially uneven distribution of the intestinal ENS.

Conclusion: CVS-IFOM may be supportive for intraoperative pathological diagnosis during surgeries in Hirschsprung disease.