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Updated: Jun 13, 2025
Immunostaining to Visualize Murine Enteric Nervous System Development
Published on: April 29, 2015
Dynamic Pathology of Enteric Neural Network Using Curcumin-assisted Multiphoton Laser Imaging in Hirschsprung Disease
Yuhki Koike1, Akira Mizoguchi2,3, Keiichi Uchida4
1Departments of Gastrointestinal and Pediatric Surgery, Mie University Graduate School of Medicine, Tsu, Japan.
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.
Insights
A new method, curcumin vital staining combined with intravital fluorescent observation (CVS-IFOM), allows detailed real-time analysis of the enteric nervous system (ENS). This technique aids in diagnosing conditions like Hirschsprung disease by visualizing microscopic tissue details without damage.
Area of Science:
- Microscopy and imaging techniques
- Gastrointestinal physiology
- Neuroscience
Background:
- Microscopic observation of living tissue is challenging due to potential damage.
- A novel intravital fluorescent observation method (IFOM) was developed, combining multi-photon laser scanning microscopy (MPLSM) with curcumin vital staining (CVS-IFOM).
Purpose of the Study:
- To analyze the enteric nervous system (ENS) in mice and human patients with hypoganglionosis and Hirschsprung disease using CVS-IFOM.
- To evaluate the efficacy of CVS-IFOM in visualizing ENS structures compared to conventional methods.
Main Methods:
- A viability study compared live ENS images from curcumin-stained mice and GFP mice using MPLSM.
- CVS-IFOM was applied to examine resected colon tissues from patients with hypoganglionosis and Hirschsprung disease.
Main Results:
- Detailed ENS histological features were observed in curcumin-stained mice.
- CVS-IFOM revealed ENS details in hypoganglionosis patients not visible with H&E or calretinin immunohistochemistry.
- In Hirschsprung disease, CVS-IFOM demonstrated gradual ENS hypoplastic changes and circumferentially uneven distribution.
Conclusions:
- CVS-IFOM provides detailed visualization of the ENS in living tissue.
- This method supports intraoperative pathological diagnosis for conditions like Hirschsprung disease.
