Cell Tissue Res (2012) 347:319–326 DOI 10.1007/s00441-011-1312-5
REGULAR ARTICLE
Ultrastructural characterization of interstitial cells of Cajal associated with the submucosal plexus in the proximal colon of the guinea pig Hiromi Tamada & Terumasa Komuro
Received: 13 September 2011 / Accepted: 20 December 2011 / Published online: 31 January 2012 # Springer-Verlag 2012
Abstract Interstitial cells of Cajal (ICC) associated with the submucosal (submucous) plexus (ICC-SP) in the proximal colon of the guinea pig were studied by immunohistochemistry and electron microscopy. Whole-mount stretch preparations with c-Kit immunohistochemistry revealed that a number of ICC-SP constituted a dense cellular network around the submucosal plexus. Some of these ICC-SP were observed in the vicinity of the muscularis mucosae in sections immunostained for c-Kit and α-smooth muscle actin. Ultrastructural observation demonstrated, for the first time, that ICC-SP of the proximal colon of the guinea pig retained typical ultrastructural characteristics of ICC repeatedly reported in association with the tunica muscularis of the gastrointestinal tract: a basal lamina, caveolae, many mitochondria, abundant intermediate filaments and the formation of gap junctions with the same type of cells. The most remarkable ultrastructural finding was the presence of thick bundles composed of the processes of ICC-SP connected to each other via large gap junctions. These ICC-SP might be involved in the main mucosal functions of the proximal colon of the
This study was supported by Grant-in-aid for JSPS Fellows from the Ministry of Education, Culture, Sports and Technology, Japan (23•4352 to H.T.). H. Tamada : T. Komuro (*) Laboratory of Histology and Neuroscience, Department of Health Science and Social Welfare, Faculty of Human Sciences, Waseda University, 2-579-15 Mikajima, Tokorozawa, Saitama 359-1192, Japan e-mail:
[email protected] H. Tamada Japan Society for the Promotion Science, 8 Ichiban-cho, Chiyoda-ku, Tokyo 102-8472, Japan
guinea pig, namely the transportation of water and electrolytes, possibly via their involvement in the spontaneous contractions of the muscularis mucosae. Keywords Interstitial cells of Cajal . c-Kit . Submucosal plexus . Proximal colon . Ultrastructure . Guinea pig
Introduction Interstitial cells of Cajal (ICC) play essential roles in gastrointestinal motility. ICC associated with the myenteric plexus (ICC-MP) act as a pacemaker for peristaltic contractions and ICC located within the muscle layer (ICC-IM) act as intermediates in neuromuscular transmission (Sanders 1996; Huizinga 1999; Ward and Sanders 2001). In addition, ICC have been found in tissues that have no relationship to the tunica muscularis for gastrointestinal motility and have long puzzled researchers with regard to their functions. Such ICC lie in the subserosal layer (ICC-SS; Aranishi et al. 2009) and are associated with the submucosal (submucous) plexus (ICC-SP; Kunisawa and Komuro 2008; MiyamotoKikuta et al. 2009; Tamada and Komuro 2011). ICC-SP of the stomach (Kunisawa and Komuro 2008) and caecum of guinea pigs (Tamada and Komuro 2011) have been proposed to be involved with the regulation of specific mucosal functions, such as the secretion, absorption and transport of fluids. In this context, ICC-SP might have a certain fundamental role related to the mucosa, although their distribution in other regions has not as yet been elucidated, despite the submucosal plexus being distributed throughout the gastrointestinal tract from the esophagus to the anus. The proximal colon of the guinea pig is active in the absorption of water and electrolytes (Araki et al. 1996;
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Skinner and O'Brien 1996) and is the site of the microbial fermentation of carbohydrates, which is vital for the production of short chain fatty acids (Stevens 1977; Argenzio 1985; Oltmer and von Engelhardt 1994). As ICC-SP have been found in the caecum of the guinea pig, a structure that has similar functions to the proximal colon, we can expect the presence of ICC-SP in the proximal colon of this animal. The demonstration of ICC-SP in the proximal colon based on such an assumption would represent another step in the elucidation of the physiological significance of ICC-SP. Further, the distribution of ICC-SP has been immunohistochemically confirmed in the above-mentioned tissues of the guinea pig but no studies have reported the ultrastructure of ICC-SP. The ultrastructural features of ICC-SP might also provide clues regarding their functional significance. Thus, in the present study, we have sought to reveal the distribution of ICC-SP in the submucosal layer of the proximal colon of the guinea pig via c-Kit immunohistochemistry and to clarify their ultrastructural characteristics.
Materials and methods Animals and tissue preparation The female Hartley guinea pigs (6 weeks old) were dissected under terminal anesthesia with ether. All procedures were performed in accordance with the guidelines for the care and use of laboratory animals of the Faculty of Human Sciences of Waseda University. The portion of the colon chosen for the present study was approximately 5 cm long, beginning approximately 2 cm from the junction between the caecum and colon.
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20 min to prevent non-specific reactivity and then incubated with monoclonal rat anti-mouse CD117 antibody (c-Kit; 1:200; eBioscience, San Diego, Calif., USA) to label ICC, rabbit anti-human PGP9.5 antibody (1:500; Ultraclone, Yarmouth, UK) to label the nervous component and mouse anti-α-smooth muscle actin antibody (1:100; Sigma Aldrich, St. Louis, Mo., USA) to label smooth muscle cells. Next, specimens were incubated with secondary antibodies conjugated with either fluorescein isothiocyanate (goat antirat IgG; 1:200; Molecular Probes, Eugene, Ore., USA), tetramethylrhodamine isothiocyanate (swine anti-rabbit IgG; 1:200; Dako, Glostrup, Denmark), or Cy3 (goat antimouse IgG; 2.5μg/ml; Millipore, Billerica, Mass., USA). Specimens were observed with a confocal laser scanning microscope (Leica TCS SP2, Leica Microsystems, Wetzlar, Germany). All antibodies were diluted in PBS containing 2% bovine serum albumin. Electron microscopy Short segments of the proximal colon were placed in a fixative containing 3% glutaraldehyde and 4% paraformaldehyde in 0.1 M cacodylate buffer, pH 7.4, for 1 h at 4 C. The specimens were then rinsed in the same buffer and postfixed in 1% osmium tetroxide in the same buffer for 2 h at 4°C, rinsed with distilled water, block-stained overnight in a saturated solution of uranyl acetate, dehydrated in an ethyl alcohol series, and embedded in epoxy resin. Following examination of semi-thin sections stained with toluidin blue to select suitable areas, ultrathin sections were cut by using an ultramicrotome (Leica Microsystems), double-stained with uranyl acetate and lead citrate and processed for observation with a transmission electron microscope (JEOL JEM 1200EX II, JEOL, Tokyo, Japan).
Immunohistochemistry Short segments of the colon were removed, briefly rinsed in 0.1 M phosphate-buffered saline (PBS), fixed for 20 min in acetone at 4°C and then washed in PBS. To obtain cryosections, the sections were cut into small pieces, immersed in sucrose solutions, embedded in OCT compound (Sakura Finetek, Torrance, Calif., USA) and frozen in liquid nitrogen. Cryosections with a thickness of 14μm were obtained by using a cryostat (Carl Zeiss MicroImaging, Göttingen, Germany) and mounted on MAS-coated slide glasses (Matsunami Glass Industries, Osaka, Japan). To produce whole-mount stretch preparations, the longitudinal muscle layer was peeled off and the mucosal layer was scraped off under a dissection microscope. Isolated submucosal layers with the circular muscles were placed in PBS containing 0.3% Triton X-100 for 20 min. Specimens were subsequently pre-incubated in 4% Block Ace solution (Dainippon Seiyaku, Osaka, Japan) for
Results Various tissue layers of the proximal colon of the guinea pig could be distinguished in cryostat sections observed by using Nomarski optics, including the muscularis mucosae, submucosa, circular muscle layer, longitudinal muscle layer and serosa (Fig. 1a). Specific distributions of c-Kit-immunoreactive ICC were clearly observed in fluorescent micrographs of the same sections (cf. Fig. 1a, b). The circular and longitudinal muscle layers contained ICC-CM and ICC-LM, respectively. Between the two muscle layers, ICC-MP were seen around the myenteric ganglia. Strong immunoreactivity of ICC associated with the submuscular plexus (ICC-SMP) clearly contoured the inner surface of the circular muscle layer. Thus, the submucosa was identified as a space between a wavy line demarcated by ICC-SMP and a dark band of the
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Fig. 1 a Longitudinal cryostat section of the proximal colon of the guinea pig observed by Nomarski optics. The thick circular muscle layer (cm) is outwardly adjacent to the longitudinal muscle layer (lm) and inwardly facing the submucosa (sm) along a wavy line (arrows). The narrow band with a homogeneous appearance above the submucosa is the muscularis mucosae (mm). Bar 80μm. b Same section as in a but stained with c-Kit (green) and PGP9.5 (red) antibodies. The tissue space of the submucosa is identified between a dark band of the muscularis mucosae (mm) and the wavy line of interstitial cells of Cajal (ICC) associated with the submuscular plexus (ICC-SMP; arrowheads), which demarcates the inner border of the circular muscle layer. Many c-Kit immunoreactive ICC-SP are observed within this space; some are present in the vicinity of the submucosal ganglion (sp). ICCCM (cm) and ICC-LM (lm) were found within the circular and longitudinal muscle layers, respectively. ICC-MP were observed around the myenteric ganglion (mp). Bar 80μm. c Longitudinal section stained with c-Kit (green) and α-smooth muscle actin (red) antibodies. The muscularis mucosae (mm) is identified as a narrow band exhibiting α-
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smooth muscle actin immunoreactivity above the circular (cm) and longitudinal (lm) muscle layers. Many c-Kit-immunoreactive ICC-SP in the submucosa are partly observed in the vicinity of the muscularis mucosae (arrow). Bar 40μm. d Higher magnification revealing the relationship between ICC-SP (sp) and the muscularis mucosae (mm). Bar 10μm. e Whole-mount stretch preparation of the submucosal layer stained with c-Kit (green) and PGP9.5 (red) antibodies. Well-developed networks of ICC-SP have a layered arrangement within the tissue depth of the submucosa in the vicinity of the network of the submucosal plexus containing ganglia (star). Bar 80μm. f Higher magnification of the same ganglion (star) as in e to reveal the details of the cell shape of ICC-SP. The cell bodies of ICC-SP are triangular (arrows) and often project approximately three processes. Long processes of ICC-SP appear to connect with each other. Bar 20μm. g Hiher magnification of the aggregation of the processes of ICC-SP. The central part of this bundle (arrow) appears to be composed of many processes derived from various sources (arrowheads), although the resolution of the fluorescent micrograph is not sufficient to determine their origin. Bar 20μm
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muscularis mucosae that contained only a few nerves (Fig. 1b). Sectioned profiles of the ganglia and nerve bundles of the submucosal plexus were scattered in this space. A number of c-Kit-immunoreactive cells or ICC-SP were densely observed in the vicinity of the plexus. Staining of these sections with both the anti-c-Kit antibody and the antiα-smooth muscle actin antibody revealed that some of these ICC-SP lay adjacent to the muscularis mucosae (Fig. 1c, d). However, no ICC were observed within the layer of the muscularis mucosae. In whole-mount stretch preparation, abundant ICC-SP were observed as the c-Kit-immunoreactive dense network in the superimposition of the network of the submucosal plexus (Fig. 1e). The network of ICC-SP extended at the tissue plane of the submucosal plexus and also at various tissue depths within the submucosa. This layered arrangement was confirmed in the three-dimensional reconstruction of ICC-SP (data not shown). These networks appeared to connect with each other between the various tissue layers. The cell bodies of ICC-SP were usually triangular and measured approximately 20μm in diameter (Fig. 1f). They often projected three long processes connecting to each other. Of particular note, these processes appeared to form bundles or an aggregated structure (Fig. 1g). ICC-SP made many contacts with each other, occurring both with the cell bodies and with the processes. Since the networks of ICC-SP had no particular axis, these could easily be distinguished from the network of ICC-SMP, which showed a longer axis roughly parallel with the running direction of the circular muscle cells. Structural relationships between the networks of ICC-SP and ICCSMP had not been detected to date. Via electron microscopy, ICC-SP were identified as elongated cells surrounding the submucosal ganglion consisting of neurons and glial cells, although thin processes in fibroblasts often intervened (Fig. 2a, b). In addition, they were also observed in the wide connective tissue space that was far from the submucosal ganglia (Fig. 3a). They were easily distinguished from the fibroblasts, which contained welldeveloped granular endoplasmic reticulum. ICC-SP were ultrastructurally characterized by the presence of caveolae, basal lamina, many mitochondria, abundant intermediate filament, subsurface cisternae and the formation of gap junctions with the same cell type (Figs. 2c, 3b, c). ICC-SP and their processes were observed in the close vicinity of nerve bundles and blood vessels but close contacts between ICC-SP and nerve varicosities were rarely observed (Fig. 3d). The most conspicuous feature of ICC-SP as demonstrated by ultrastructural observation was the formation of bundlelike structures composed of processes of ICC-SP (Fig. 4a). These were connected to each other by gap junctions and were surrounded by a loose sheath of fibroblasts. In the
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profile in Fig. 4a, eight of 15 processes were connected with each other at seven sites via large gap junctions. The total area of the processes connected via the gap junctions (eight processes) was approximately two-thirds of the total sum of all processes (15) in this profile. Similar but thinner bundles of ICC-SP were observed in the narrow spaces between the large venules (Fig. 4b). Notably, in the profile in Fig. 4b, a long gap junction (about 500 nm) was observed along the axis of slender processes, whereas a complete circular gap junction encompassed a penetrating process that was probably joined to this bundle from the perpendicular direction (Fig. 4c). Aggregated structures of the cell bodies of ICC-SP were not observed. Sectioned profiles of thin bundles of ICC-SP were widely observed in the connective tissue space without any particular connection with other structures. We attempted to elucidate ultrastructural evidence of the close relationship between ICC-SP and the muscle cells of the muscularis mucosae but no such structure has been obtained, so far. Their minimum distance measures about 1.5μm.
Discussion The present study has revealed the ultrastructural features of ICC-SP for the first time. These cells are characterized by the presence of basal lamina, caveolae, many mitochondria, abundant intermediate filaments and large gap junctions connected with each other. Thus, ICC-SP can be clearly distinguished from the fibroblasts and from so-called telocytes that have been recently proposed as a new type of interstitial cell in the human gut but which does not have a distinct basal lamina (Pieri et al. 2008; Popescu and Faussone-Pellegrini 2010). These features are comparable with those of typical, most muscle-like ICC (Type 3) mainly observed in relationship with the tunica muscularis (Komuro et al. 1999) and are similar to those of ICC-SMP of the colon of the guinea pig (Ishikawa and Komuro 1996). However, unlike ICC-SMP, ICC-SP rarely exhibit close contacts with nerve varicosities. Their close contacts with the muscle cells of the muscularis mucosae have not been observed, so far. Thus, an intermediary role of ICC-SP in neuromuscular transmission is unlikely. In contrast to the ICC-SP of the stomach (Kunisawa and Komuro 2008), ileocaecal junction (Miyamoto-Kikuta et al. 2009) and caecum (Tamada and Komuro 2011), ICC-SP and their processes in the proximal colon have been frequently observed in wide connective tissue spaces away from the submucosal ganglia. Basket-like structures composed of multipolar ICC-SP have been observed around the submucosal ganglia in the caecum of the guinea pig but these structures have not been observed in the proximal colon.
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Fig. 2 a Electron micrograph showing the submucosal ganglion consisting in neurons (N) and glial cells (G) surrounded by thin processes of ICC-SP (arrows) intermingling with the processes of fibroblasts. Bar 8μm. b ICC-SP (star) located in the vicinity of the submucosal ganglion. The spindle-shaped cell body of ICC-SP containing an
elongated nucleus lies over the thin processes of the fibroblasts (fb) from the ganglion (SG). Bar 2μm. c Higher magnification of the left half of the cytoplasm of the same ICC-SP as shown in b. Caveolae (arrowhead) and many mitochondria (m) are observed in the cytoplasm. Bar 1μm
In this respect, ICC-SP of the proximal colon are different from ICC-SP reported in other regions hitherto. ICC-SP have not been detected in the small intestine of the guinea pig in our preliminary experiments. The most remarkable feature of ICC-SP is the presence of bundles composed of many processes of ICC-SP. These processes are connected with each other by numerous large gap junctions and, thus, can be regarded as a type of functional syncytium. In our quantitative analysis of the bundles,
the total area of processes connected via gap junctions comprises approximately two-thirds of the sum of all processes in the bundle. Because no cell bodies have been observed in the bundles, these structures probably represent a profile of cables rather than large cell masses. The wide distribution of the slender processes of ICC-SP, including those located near large blood vessels, which also exhibit gap junction connections, might have continuity with the large bundles and constitute a type of plexus via the
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Fig. 3 a ICC-SP (ic) and fibroblast (fb) in the submucosal connective tissue occupied by abundant collagen fibrils (co). Note the welldeveloped granular endoplasmic reticulum (er) in the cytoplasm of the fibroblast. Bar 3μm. b Higher magnification of the same ICC-SP as shown in a. Gap junctions are observed between this cell and the processes of the same type of cells (arrow) and between the other two processes (double arrowhead). Caveolae, mitochondria and
intermediate filaments (arrowheads) are observed. Bar 750 nm. Inset Basal lamina (arrowheads) is clearly visible along the cell membrane of the ICC-SP. Bar 120 nm. c Higher magnification of the gap junction indicated by arrow in b. Bar 80 nm. d Close contact between a nerve varicosity containing cored vesicles and the process of an ICC-SP that is characterized by many intermediate filaments (star). Bar 500 nm
processes of ICC-SP. This assumption can be justified by the immunohistochemical observation of a complex network of c-Kit-immunoreactive cells in the submucosal connective tissue space. Although no relevant experiments have been carried out that allow us to suggest the functional significance of these structures, networks of ICC-SP including thick cable-like structure appear to have a potential importance for activities in the proximal colon. Indeed, the presence of a dense
cellular network of ICC-SP in the submucosal layer of the proximal colon of the guinea pig indicates that ICC-SP are involved in functions similar to those of the submucosal plexus, which plays a role in the regulation of mucosal and submucosal activities, such as absorption, secretion and transportation of fluids (Furness 2006), although no direct experiments have revealed the functional role of ICC-SP. In this context, the contractions and relaxation of the muscularis mucosae change both the surface area of the
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Fig. 4 a Bundle-like structure consisting processes of ICC-SP. The processes contain abundant intermediate filaments and mitochondria and have a basal lamina (double-headed arrows). They are connected with each other via gap junctions (arrows), thereby constituting the large bundle. This bundle is surrounded by loose sheaths of fibroblasts (fb). The longer diameter of this bundle measures about 6μm and the shorter diameter measures about 3μm. Eight processes of 15 processes make contacts via each other with gap junctions. The total area of the processes connected via gap junction (8μm2) comprises approximately
two-thirds of the total sum area of all 15 processes (13μm2). The total sum of the length of the seven gap junctions is approximately 3μm. Bar 500 nm.b Slender processes of ICC-SP (ic) located in the narrow space between two large venules containing red corpuscles. Bar 3μm. c Higher magnification of the ICC-SP corresponding to the area of b confirming that these are connected by large gap junctions (arrow). One process containing mitochondria, which probably joined this small bundle from the perpendicular direction, is completely encircled by a gap junction (double-headed arrow). Bar 500 nm
mucosa and the space capacity of the submucosa and, in turn, affect the flow of fluids. Tetrodotoxin-resistant spontaneous contractions of the muscularis mucosae have been observed in the rabbit stomach (Percy et al. 1999, 2002) and these contractions have been considered responsible for oscillations in glandular pressure and in blood velocity in the mucosa (Synnerstad et al. 1998). Tetrodotoxin-resistant slow spontaneous contractions of the muscularis mucosae have also been reported in the proximal
colon of rabbits (Percy et al. 1992) and guinea pigs (Ishikawa and Ozaki 1997). Although no specific contact between ICCSP and the muscularis mucosae has been observed at the ultrastructural level in the present study, ICC-SP are often observed in the vicinity of the muscularis mucosae in immunohistochemical preparations. ICC-SP located beneath the musuclaris mucosa might be involved in the regulation of their spontaneous movement as a pacemaker, as suggested in the stomach of the guinea pig (Kunisawa and Komuro 2008).
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Further, some doubt occurs regarding the pacemaker of the main muscle coat (the tunica muscularis) of the colon. ICC-SMP have been widely acknowledged as the primary pacemaker, together with ICC-MP as the secondary pacemaker. Preparations created after removal of the submucosa do not exhibit slow waves (Smith et al. 1987; Plujà et al. 2001) or rhythmic contraction (Yoneda et al. 2004; Kodama et al. 2010) attributable to ICC-SMP. The present observations clearly demonstrate that ICCSP are located in the connective tissue space and are thus a different category of cells from ICC-SMP that are closely adherent to the inner border of the circular muscle layer. However, the presence or absence of submucosa also clearly influences the results of these physiological experiments. Thus, some room exists for a re-examination of these results in the light of the finding that the submucosa contains dense networks of ICC-SP. The involvement of ICCSP cannot be ruled out in these experiments, although uncertainties remain as to whether dog, rat and mouse colons possess a similar dense distribution of ICC-SP and whether ICC-SP have a functional relationship with ICCSMP. However, the reason(s) for the dominant pacemaker site of the colon being located at the submucosal border of the circular muscle layer, unlike those of the stomach and small intestine, should be considered. In conclusion, the present study has demonstrated welldeveloped networks of ICC-SP in the vicinity of the submucosal plexus and revealed their ultrastructural features for the first time, thus providing important clues for interpreting functional experiments and for future studies.
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