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

Abstract

BACKGROUND: In living tissue, it has been difficult to make microscopic-level observations without damaging the tissue. We have invented a novel intravital fluorescent observation method (IFOM) for real-time tissue observation, combining multiphoton laser scanning microscopy 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 (HSCR). METHODS: In an initial viability study, we compared live ENS images from nonfluorescent C57BL6 mice stained with curcumin (n = 5) and green fluorescent protein mice (n = 5) using multiphoton laser scanning microscopy. We then explored CVS-IFOM for the live examination of resected colon tissues from 1 patient with hypoganglionosis and 3 patients with HSCR. RESULTS: In the viability study, detailed ENS histologic features were only observed in the curcumin-stained mice. In the patient with hypoganglionosis, CVS-IFOM provided ENS details that were not visualized under hematoxylin and eosin staining or calretinin immunohistochemistry, allowing the analysis of ENS size, neural bundle number, and neural cell number per plexus. In patients with HSCR, CVS-IFOM showed a gradual hypoplastic change in the ENS from the oral edge to the anal edge, detecting disproportionate changes in the ENS within the same intestinal level, supporting a circumferentially uneven distribution of the intestinal ENS. CONCLUSIONS: CVS-IFOM may be supportive for intraoperative pathologic diagnosis during surgeries for HSCR.