Multiphoton microscopy to identify and characterize the transition zone in a mouse model of Hirschsprung disease.

ABSTRACT

BACKGROUND: The distribution of ganglion cells in the transition zone of Hirschsprung Disease (HD) colons is extremely variable. Determining the resection margin based on intraoperative biopsies may be imprecise. Multiphoton microscopy (MPM) is a novel imaging technology with the ability to visualize tissues in real time. In this study, we evaluate the potential of MPM to quantify ganglion cells in a murine model of HD. METHODS: After IACUC approval, formalin-fixed colons from 7 wild type (WT) and 6 Endothelin Receptor B gene (EdnrB) homozygous knockout (KO) mice with distal colonic aganglionosis were assessed by MPM for the presence of myenteric ganglion cells. MPM images were captured starting from the anus progressing proximally at 5mm intervals. Hematoxylin and eosin (H&E) stained biopsies of the imaged were correlated with MPM findings. RESULTS: WT specimens showed normal myenteric plexus ganglia throughout the examined colon. In contrast, distal colons of EdnrB KO animals were devoid of ganglia up to 10mm from the anus. Ganglion cells were visible starting at 20-30 mm proximal to the anus. The density of ganglion cells seen by MPM and histology correlated well. CONCLUSIONS: MPM can clearly identify the myenteric plexus ganglia in both WT and KO mouse colons. Comparison with the H&E-stained sections showed reproducible correlation. MPM-based real-time imaging of the myenteric plexus may become a useful intraoperative decision-making tool in the future.