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Relationship between interstitial cells of Cajal, fibroblast-like cells and inhibitory motor nerves in the internal anal sphincter

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Abstract

Interstitial cells of Cajal (ICC) have been shown to participate in nitrergic neurotransmission in various regions of the gastrointestinal (GI) tract. Recently, fibroblast-like cells, which are positive for platelet-derived growth factor receptor α (PDGFRα+), have been suggested to participate additionally in inhibitory neurotransmission in the GI tract. The distribution of ICC and PDGFRα+ cell populations and their relationship to inhibitory nerves within the mouse internal anal sphincter (IAS) are unknown. Immunohistochemical techniques and confocal microscopy were therefore used to examine the density and arrangement of ICC, PDGFRα+ cells and neuronal nitric-oxide-synthase-positive (nNOS+) nerve fibers in the IAS of wild-type (WT) and W/W v mice. Of the total tissue volume within the IAS circular muscle layer, 18% consisted in highly branched PDGFRα+ cells (PDGFRα+-IM). Other populations of PDGFRα+ cells were observed within the submucosa and along the serosal and myenteric surfaces. Spindle-shaped intramuscular ICC (ICC-IM) were present in the WT mouse IAS but were largely absent from the W/W v IAS. The ICC-IM volume (5% of tissue volume) in the WT mouse IAS was significantly smaller than that of PDGFRα+-IM. Stellate-shaped submucosal ICC (ICC-SM) were observed in the WT and W/W v IAS. Minimum surface distance analysis revealed that nNOS+ nerve fibers were closely aligned with both ICC-IM and PDGFRα+-IM. An even closer association was seen between ICC-IM and PDGFRα+-IM. Thus, a close morphological arrangement exists between inhibitory motor neurons, ICC-IM and PDGFRα+-IM suggesting that some functional interaction occurs between them contributing to inhibitory neurotransmission in the IAS.

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Acknowledgements

The authors acknowledge the following individuals for their technical assistance with this work: Maria Durazo (Nevada State Health Laboratory), Byoung Koh, and Yulia Bayguinov.

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Authors and Affiliations

  1. Department of Physiology and Cell Biology, University of Nevada School of Medicine, Reno, NV 89557, USA

    Caroline A. Cobine, Grant W. Hennig, Masaaki Kurahashi, Kenton M. Sanders, Sean M. Ward & Kathleen D. Keef

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  1. Caroline A. Cobine
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  2. Grant W. Hennig
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  3. Masaaki Kurahashi
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  4. Kenton M. Sanders
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  5. Sean M. Ward
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  6. Kathleen D. Keef
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Corresponding author

Correspondence to Kathleen D. Keef.

Additional information

This work was supported by grants DK078736 to K.D.K., DK057236 to S.M.W. and COBRE P20RR018751 to G.W.H. and by grant PPG DK41315. Imaging analysis was performed in a Core laboratory supported by P20RR018751.

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Supplementary Figure 1

Anaglyphs of intramuscular interstitial cells of Cajal (ICC-IM) and PDGFRα+-IM in the circular muscle of the mouse IAS. Colored glasses are necessary to view the three-dimensional nature of these cells (red for the left eye/cyan for the right eye). Note the striking differences in cell branching and interconnectivity of PDGFRα+-IM (b) versus ICC-IM (a). Images derived from the same confocal stack as in Fig. 8a. Optical section thicknesses: a 8.75 μm, b 8.75 μm (GIF 466 KB)

High resolution image file (TIF 13.3 mb)

Supplementary Figure 2

Enhanced green fluorescent protein (eGFP) is present in the cell body of every PDGFRα+ cell and PDGFRα is present in eGFP+ cell bodies. Cryostat sections from the internal anal sphincter (IAS) of the Pdgfra tm11(eGFP)Sor /J heterozygote mouse were labeled with a PDGFRα antibody (red) and cell nuclei were identified by the DNA counterstain 4,6-diamidino-2-phenylindole (DAPI; blue) as in Fig. 5. a–c Labeling within the circular muscle (CM) layer including the myenteric (My) surface. d–f Labeling within the longitudinal muscle (LM) layer. PDGFRα, eGFP (green) and DAPI are all shown in a, d, whereas DAPI has been omitted in b, e, and only PDGFRα is shown in c, f. b Even PDGFRα+ cells with predominant DAPI labeling in the cell body also express eGFP (star). f eGFP+ cells with little surrounding PDGFRα labeling contain PDGFRα within the cell body (white triangle). Optical section thicknesses: a-c 6.5 μm, d-f 10.5 μm (GIF 329 KB)

High resolution image file (TIF 473 kb)

Supplementary Figure 3

Distribution of nerve fibers positive for neuronal nitric oxide synthase (nNOS+) is similar in IAS of the wild-type (WT) and W/W v mouse. Various populations of nNOS+ nerve fibers are found in the IAS of the WT and W/W v mouse. nNOS+ nerve fibers along the submucosal (SM) surface form a neural plexus (a, d); intramuscular (IM) nerve fibers run parallel to the smooth muscle cells (b, e) and form a plexus at the myenteric (My) surface where cell bodies are present (c, f). Optical section thicknesses: a 3.5 μm, b 4.5 μm, c 5 μm, d 7.5 μm, e 9 μm, f 10 μm (GIF 474 KB)

High resolution image file (TIF 473 kb)

Supplementary Figure 4

Revolving video of ICC-IM and PDGFRα+-IM. The three-dimensional relationship of ICC-IM (red) to PDGFRα+-IM (green), as shown in Fig. 8a, is apparent (MPG 13810 kb)

Supplementary Figure 5

Rotation of one cell type within dual-labeled images results in a decrease in alignment between cell types. Alignment of ICC-IM vs. nNOS+ nerve fibers (a, squares), PDGFRα+-IM vs. nNOS+ nerve fibers (b, triangles) and ICC-IM vs. PDGFRα+-IM (c, circles) are compared in unmodified images (open symbols) versus images in which one cell type has been rotated by 90° with respect to the other (closed symbols). All curves show a decrease in maximum and in slope. The decline in alignment seen after rotation is greatest for ICC-IM vs. PDGFRα+-IM (c) indicating that these cells are preferentially aligned with one another in the CM axis (GIF 36.5 KB)

High resolution image file (TIF 9.09 kb)

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Cobine, C.A., Hennig, G.W., Kurahashi, M. et al. Relationship between interstitial cells of Cajal, fibroblast-like cells and inhibitory motor nerves in the internal anal sphincter. Cell Tissue Res 344, 17–30 (2011). https://doi.org/10.1007/s00441-011-1138-1

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