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. 2012 May 15;302(10):G1099-110.
doi: 10.1152/ajpgi.00432.2011. Epub 2012 Mar 15.

3-D illustration of network orientations of interstitial cells of Cajal subgroups in human colon as revealed by deep-tissue imaging with optical clearing

Yuan-An Liu  1 , Yuan-Chiang Chung, Shien-Tung Pan, Yung-Chi Hou, Shih-Jung Peng, Pankaj J Pasricha, Shiue-Cheng Tang
Affiliations

3-D illustration of network orientations of interstitial cells of Cajal subgroups in human colon as revealed by deep-tissue imaging with optical clearing

Yuan-An Liu et al. Am J Physiol Gastrointest Liver Physiol. .

Abstract

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.

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Figures

Fig. 1.
Fig. 1.
Optical clearing of the human colon wall and c-kit immunostaining. A and B: optical clearing promotes photon penetration across the specimen of the human colon wall. The microstructures and their associated interstitial cells of Cajal (ICC) subgroups are listed in the figure. MY, myenteric plexus; LM, longitudinal muscles; CM circular muscles; SM, sumucosal boundary. Thickness: 400 μm. C–F: immunostaining acquired from 2 sources of c-kit antibody. C and D: company MBL; E and F: company Epitomics. The vertical pixel line in the middle of D (1,024×1,024 pixels) correlates to the vertical axis of the signal intensity panel (right), which indicates the signal profile of the pixel line (25). D and F follow the same arrangement. Between the 2 sources, Epitomics' antibody generates less background noises, leading to higher signal-to-noise ratios (i.e., higher image quality) to identify the processes of ICC in the projection (E).
Fig. 2.
Fig. 2.
Three dimensional (3-D) morphology of ICC-MY and their association with ICC-LM and ICC-CM. A: representative 2-dimensional (2-D) micrograph of an image stack acquired at the myenteric plexus. The c-kit (red) and nuclear (green) signals are merged with the transmitted light micrograph to reveal the ICC and their associated microstructures. Arrows indicate the periganglionic ICC-MY. The serial optical sections of the scanned volume are shown in Supplemental Video 1. B and C: merged and individual 3-D projections of the c-kit (gray) and nuclear (green) signals shown in Supplemental Video 1. D: projections of C from 3 different points of views. The cyan arrow indicates the orientation of the ICC-CM, which is discussed in Fig. 4. E and F: 2 additional examples of ICC at the myenteric plexus. The projections show a joint network arrangement of ICC-MY with ICC-LM and ICC-CM. A 360-degree panoramic view of E is shown in Supplemental Video 2.
Fig. 3.
Fig. 3.
ICC-LM in the longitudinal muscle. A: optical clearing reveals the texture of the longitudinal muscle. Arrows indicate two interfaces between the muscle segments, which correlate to 2 of the ICC-LM strata shown in E. B: representative 2-D micrograph of the c-kit (red) and nuclear (green) signals in the longitudinal muscle. Arrows indicate the cell bodies of ICC. The serial optical sections of the scanned volume are shown in Supplemental Video 3. C and D: merged and individual 3-D projections of the ICC-LM (gray) and nuclei (green) shown in Supplemental Video 3. E and F: volume-edited projections of c-kit signals reveal the orientations of the ICC-LM. A cuboid of the c-kit projection was digitally subtracted from the center of the scanned volume to expose the orientations of the ICC-LM strata which line the boundaries of the muscle segments (arrows in A) and extend toward the serosal and the longitudinal directions. A 360-degree rotation of the projection is shown in Supplemental Video 4.
Fig. 4.
Fig. 4.
Repetitive and organized ICC-CM layers in the circular muscle. A and B: merged and individual 3-D projections of the ICC-CM (gray) and nuclei (green) in the transverse circular muscle section. A 360-degree panoramic view of B is shown in Supplemental Video 5. C: 90-degree rotation of B shows the repetitive and organized ICC-CM. Arrows indicate that the ICC strata extend toward the submucosa from the myenteric plexus in addition to running parallel to the circular muscle axis (as revealed in A). D–F: 3-D imaging of ICC-CM in the longitudinal section of the circular muscle. D: the orientation of muscles as revealed by transmitted light imaging with optical clearing. E: 3-D projection of ICC-CM. F: merged projection of E with a 2-D micrograph of smooth muscle actin and nuclear signals. A 360-degree panoramic view of F is shown in Supplemental Video 6. D–F are derived from the same image stack.
Fig. 5.
Fig. 5.
Projection of a layer of ICC-CM in space. A layer of c-kit signals and their associated nuclei were segmented from the image stack shown in Fig. 4A for panoramic visualization of the ICC-CM. A and B: 2 projection angles. The 360-degree rotation of the segmented plexus is shown in Supplemental Video 7.
Fig. 6.
Fig. 6.
A layer of ICC-SM at the submucosal boundary. A: merged display of the transmitted light micrograph with c-kit (red) and nuclear (green) signals at the boundary between the submucosa and circular muscle. The box indicates a dense layer of ICC-SM at the interface. The serial optical sections of the scanned volume are shown in Supplemental Video 8. B–D: 3-D projections of the c-kit and nuclear signals shown in Supplemental Video 8. B is a composite display of A merged with the projection. C rotates the projection to reveal the ICC-SM (red) lining the circular muscle border. In D, the nuclear signals were removed to reveal the ICC at the interior domain of the scanned volume. A 360-degree panoramic view of D is shown in Supplemental Video 9. E and F: 3-D projections of ICC-SM. Only the c-kit signals and their associated nuclei were segmented for presentation. All images were derived from the same image stack.
Fig. 7.
Fig. 7.
Quantitation of ICC-CM in space. A: example of a 200-μm image stack of c-kit and nuclei (see Fig. 4A) assigned for quantitative analysis. B: 2-D features of the ICC-CM (red) and nuclei (green) projected from the 3rd section of the image stack shown in A. Arrows indicate the ICC. Particularly, the nuclear signals indicate the locations of the cell body to define an ICC: c-kit+ and at least 2 processes extending from the cell body.
Fig. 8.
Fig. 8.
In-depth image projections of ICC-MY in the myenteric plexuses of the 4 individuals listed in Table 1. The variation of ICC-MY in the projections is due to their association with the myenteric ganglia, which are intrinsically irregular in space. Aggregates of ICC-MY are commonly seen, compared with the dispersed morphology of ICC-CM in the upper part of the images. The 4 images were taken and projected under the same magnification and depth.
Fig. 9.
Fig. 9.
In-depth image projection helps identify mast cells (A–D) and septal ICC (C–F). A and B: mast cells in the circular muscle. These are often seen close to the submucosa (top part of the images). The specimen was derived from subject 2 in Table 1. A is 1 of the 26 images used in projection (B). Arrows indicate the mast cells; they do not have processes extending from the cell body. Transverse colon section. C and D: mast cells and ICC around and in the septa. The specimen was derived from subject 3 in Table 1. White arrows indicate the mast cells. Their morphology is similar to that of their counterparts in A and B. In addition to the mast cells, the projection also reveals the septal ICC (cyan arrows, D) lining the septa (dotted arrow, C). Transverse colon section. E and F: imaging of the septal ICC in the longitudinal colon section. The transmitted light micrograph shows the locations of the septa (red arrows) that separate the adjacent muscle segments. The in-depth image projection reveals that the septal ICC (cyan arrows) elongate and connect with ICC-MY at the myenteric plexus and run within the septa of the circular muscle layer (white arrows). E and F were taken under the same view; the red and white arrows indicate the same locations.
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