Purpose: The distribution of ganglion cells in Hirschsprung's Disease (HD) colons is extremely variable, and determining the resection margin based on a limited number of intraoperative biopsies can be inaccurate. Multiphoton microscopy (MPM) is a recently developed non-linear imaging technology with the ability to illuminate fresh unprocessed tissues in real time with near-infrared light. In this study, we evaluate the potential and accuracy of MPM to detect and quantify ganglion cells in a murine model of HD.
Methods: IACUC approval was obtained (2011-0063).Formalin-fixed colons from homozygous knockout (KO) mice for the endothelin receptor type B (EdnrB) gene (n=3) with resulting distal colon aganglionosis were assessed by MPM. Colons from 3 wild-type (WT) mice served as controls. Images were obtained starting from the anus going proximally up to 50 mm at 5 mm intervals. Full-thickness biopsies of the imaged areas were obtained, stained with hematoxylin and eosin (H&E), and correlated with the MPM findings. For quantitative analysis, myenteric ganglion cells were defined on MPM as located at the junction of longitudinal and circular muscular layers, with large, round nuclei and abundant cytoplasm.
Results: WT specimens showed normal myenteric plexus ganglia throughout the examined colon. In contrast, the distal bowels of KOs were completely devoid of ganglia up to 10 mm from the anus. Ganglion cells were visible starting at 20-30 mm proximal to the anus. The interim region (10-30 mm) showed scattered cells that could not always be conclusively identified as ganglion cells based on above criteria. The density of ganglion cells seen by MPM and on histologic sections correlated well at every level in both WT and KO colons (figure 1a). Quantitative analysis of ganglion cell density by MPM allowed us to characterize the transition zone and compare the extent of aganglionosis between different KO animals (figure 1b).
Conclusions: This preliminary ex vivo study indicates that MPM can clearly identify the myenteric plexus ganglia in WT and KO mouse colons. Comparison with the H&E-stained sections of the corresponding areas showed excellent correlation. Therefore, MPM-based real-time imaging of the myenteric plexus may become a useful intraoperative decision-making tool in the future. Given the development of several miniaturized multiphoton devices capable of integration into current laparoscopic instruments, we foresee translation of this imaging approach in large animal models and human clinical trials in the near future.
Figure 1a: Comparative MPM and H&E images of distal colon taken at 1, 25, and 30 mm from the anus. Arrows point to clusters of ganglion cells. Magnification: 300x for MPM and 200x for H&E.
Figure 1b: Quantitative analysis and comparison of the number of ganglion cells at increasing distance from the anus in two KO animals.