Morphological changes of interstitial cells of Cajal (ICC) have been proposed to characterize motility disorders. However, a global view of the network orientations of ICC subgroups has not been established to illustrate their three-dimensional (3-D) architectures in the human colon. In this research, we integrate c-kit immunostaining, 3-D microscopy with optical clearing, and image rendering to present the location-dependent network orientations with high definition. Full-depth colonic tissues were obtained from colectomies performed for nonobstructing carcinoma. Specimens of colon wall were prepared away from the tumor site. C-kit and nuclear fluorescent staining were used to identify the ICC processes and cell body. Optical clearing was used to generate transparent colon specimens, which led to panoramic visualization of the fluorescence-labeled ICC networks at the myenteric plexus (ICC-MY), longitudinal (ICC-LM) and circular (ICC-CM) muscles, and submucosal boundary (ICC-SM) up to 300 μm in depth via confocal microscopy with subcellular level resolution. We observed four distinct network patterns: 1) periganglionic ICC-MY that connect with ICC-LM and ICC-CM, 2) plexuses of ICC-LM within the longitudinal muscle and extending toward the serosa, 3) repetitive and organized ICC-CM layers running parallel to the circular muscle axis and extending toward the submucosa, and 4) a condensed ICC-SM layer lining the submucosal border. Among the four patterns, the orderly aligned ICC-CM layers provide an appropriate target for quantitation. Our results demonstrate the location-dependent network orientations of ICC subgroups and suggest a practical approach for in-depth imaging and quantitative analysis of ICC in the human colon specimen.

Background & AimsInterstitial cells of Cajal (ICC) closely associate with nerves and smooth muscles to modulate gut motility. In the ICC microenvironment, although the circulating hormones/factors have been shown to influence ICC activities, the association between ICC and microvessels in the gut wall has not been described. We applied three-dimensional (3D) vascular histology with c-kit staining to identify the perivascular ICC and characterize their morphologic and population features in the human colon wall.MethodsFull-thickness colons were obtained from colectomies performed for colorectal cancer. We targeted the colon wall away from the tumor site. Confocal microscopy with optical clearing (use of immersion solution to reduce scattering in optical imaging) was performed to simultaneously reveal the ICC and vascular networks in space. 3D image rendering and projection were digitally conducted to illustrate the ICC–vessel contact patterns.ResultsPerivascular ICC were identified in the submucosal border, myenteric plexus, and circular and longitudinal muscles via high-definition 3D microscopy. Through in-depth image projection, we specified two contact patterns—the intimate cell body-to-vessel contact (type I, 18% of ICC in circular muscle) and the long-distance process-to-vessel contact (type II, 16%)—to classify perivascular ICC. Particularly, type I perivascular ICC were detected with elevated c-kit staining levels and were routinely found in clusters, making them readily distinguishable from other ICC in the network.ConclusionsWe propose a new subclass of ICC that closely associates with microvessels in the human colon. Our finding suggests a functional relationship between these mural ICC and microvessels based on the morphologic proximity.