Department of Physiology and Cell Biology, University of Nevada School of Medicine, Reno, Nevada 89557
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ABSTRACT |
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Electrical and mechanical activity of the circular muscle layer in the rectoanal region of the gastrointestinal tract undergoes considerable changes in the site of dominant pacemaking activity, frequency, and waveform shape. The present study was performed to determine whether changes in the structural organization of the circular layer or in the density, distribution, and ultrastructure of interstitial cells of Cajal (ICC) could account for this heterogeneity in electrical and mechanical activities. Light microscopy revealed that the structural organization of the circular muscle layer underwent dramatic morphological changes, from a tightly packed layer with poorly defined septa in the proximal rectum to one of discrete muscle bundles separated by large septae in the internal anal sphincter. Kit immunohistochemistry revealed a dense network of ICC along the submucosal and myenteric borders in the rectum, whereas in the internal anal sphincter, ICC were located along the periphery of muscle bundles within the circular layer. Changes in electrical activity within the circular muscle layer can be partially explained by changes in the structure of the muscle layer and changes in the distribution of ICC in the rectoanal region of the gastrointestinal tract.
enteric nervous system; Kit immunoreactivity; internal anal sphincter; smooth muscle
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INTRODUCTION |
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IT IS NOW ACCEPTED that interstitial cells of Cajal (ICC) generate electrical slow waves and also serve as intermediates in enteric motor neurotransmission in the gastrointestinal (GI) tract (6, 7, 27, 28, 32). These hypotheses have been supported by a variety of experiments that included morphological and electrophysiological studies. In the canine proximal and distal colons, a dominant pacemaker activity exists along the submucosal surface of the circular muscle layer (21, 29), and a second, less dominant site exists at the level of the myenteric plexus (30). Sharp dissection and removal of the circular muscle-submucosal interface abolishes the dominant slow-wave pacemaking activity in the bulk of the circular muscle. Furthermore, electrophysiological studies on submucosal ICC (IC-SM) isolated from along this submucosal surface revealed for the first time, that these cells were both electrical excitable and generated spontaneous activity with a waveform similar to that found in intact tissues (20). The circular muscle layer possesses a gradient in membrane potential from a highly polarized potential along the submucosal surface to a less negative membrane potential along the myenteric border (29-31). The polarized membrane potential at the submucosal surface has been attributed to ionic membrane conductances specifically expressed in ICC. These conductances include a barium-sensitive inward rectifier that has been identified in ICC (9, 17). Ultrastructural examination of the canine proximal colon has revealed IC-SM along this surface and that these cells were coupled to one another and to neighboring smooth muscle cells by gap junctions (2, 41). IC-SM also populated septae that separated the circular muscle layer into discrete muscle bundles (22, 41). These septal ICC (IC-SEP) contribute to the electrical activity of the proximal colon (41).
The proximal rectum has similar electrophysiological characteristics as the proximal and distal colons. However, recordings from the proximal rectum to internal anal sphincter (IAS) revealed that this region of the GI tract has a complex arrangement of electrical activity that changed both in a proximal-to-distal directions as well as through the thickness of the circular muscle layer (25). The heterogeneity in the electrical activity in this region of the GI tract suggests that the anatomical structure of the tunica muscularis or the density and distribution of the specialized pacemaking ICC may vary in the terminal regions of the GI tract. To determine structural changes or changes in ICC, we performed a detailed morphological examination of the proximal rectum and IAS by using light and electron microscopy and immunohistochemistry by using antibodies against Kit, a receptor tyrosine kinase that is highly specific for ICC in the tunica muscularis of the GI tract (4, 5, 15, 19, 24, 34, 38).
Our results suggest that the morphological arrangement of the tunica muscularis in the rectum and IAS and the distribution of ICC within this region of the GI tract may explain the heterogeneity in the electrical activity recorded in a proximal-distal direction along the colon and across the thickness of the circular muscle layer in this region of the GI tract.
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MATERIALS AND METHODS |
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Mongrel dogs with an average age of 1 yr of both sexes were killed with pentobarbital sodium (100 mg/kg). After opening the abdomen, a 10-cm region of the terminal colon including the IAS was removed and placed in a bath of oxygenated Krebs-Ringer bicarbonate solution (KRB). For morphological studies, the proximal rectum (8 cm from the anal verge) and IAS were opened along the mesenteric border and fecal material was washed with KRB. Tissues were pinned to the silicon (Sylgard elastomer; Dow Corning, Midland, MI) base of a dissecting dish, and the mucosa and inner aspect of the submucosa were removed by sharp dissection and stretched to 110% of the original length and width (after incubation in 1 µM nifedipine for 15 min) before being fixed in an appropriate fixative for light or electron microscopy. Use and treatment of animals was approved by the Animal Use and Care Committee at the University of Nevada.
Light microscopy and immunohistochemistry. For light microscopy, the proximal rectum and IAS were isolated and fixed in a manner similar to that described above. The mucosa was removed by sharp dissection and the remaining tunica muscularis was subsequently fixed in ice-cold paraformaldehyde [4% wt/vol in 0.1 M phosphate buffer (PB)] at 4°C for 20-30 min. After fixation, tissues were washed for 30 min in PBS, 0.05 M, pH 7.4. For light microscopy studies, preparations were dehydrated through a graded series of alcohols and xylene before being embedded in paraffin wax. Sections were cut at a thickness of 5 µm and photographed by using a Leitz Diaplan microscope. Immunohistochemical studies were performed on tissues fixed in ice-cold paraformaledehyde before being dehydrated in graded sucrose solutions, embedded in Tissue Tek (Miles, IL), and frozen in liquid nitrogen. Tissue sections (8 µm) were cut by using a Leica CM3050 cryostat and were subsequently preincubated in 1% BSA in 0.1 M PBS for 1 h before being incubated with a rabbit polyclonal antibody raised against human Kit protein (Cat. no. CD117; DAKO, Kyoto, Japan; Lot No. 0D010A raised against amino acid sequence 963-976 at the COOH terminus end of the receptor; 1:50 dilution in 0.1 M PBS with 0.5% Triton X-100) at 4°C overnight. This antibody has been recently shown to specifically label canine ICC (13). Immunoreactivity was detected with FITC-conjugated secondary antibody (goat anti-rabbit 1:100; Vector Laboratories, Burlingame, CA) for 1 h at room temperature. Control tissues were prepared in a similar manner, omitting either primary or secondary antibody from the incubation solution. Tissue sections were examined with a Bio-Rad MRC 600 confocal microscope (Hercules, CA) with an excitation wavelength appropriate for FITC (494 nm). Confocal micrographs are digital composites of Z-series scans of 9 or 17 optical sections through a depth of 8 µm. Final images were constructed with Bio-Rad "Comos" software.
Electron microscopy. Strips of proximal rectum and IAS were placed in a fixative solution containing paraformaldehyde (4% wt/vol) and glutaraldehyde (3% vol/vol) made up in PB (0.1 M; pH 7.4) for 2 h at room temperature. Tissues were rinsed with the same buffer and postfixed with osmium tetroxide (1% wt/vol) for 2 h at 4°C. Tissues were then rinsed in distilled water, block stained with saturated aqueous uranyl acetate solution overnight, dehydrated through ethanol solutions, and embedded in Epon epoxy resin (Ted Pella, Redding, CA). Ultrathin sections were cut by using a Reichert ultramicrotome and double stained with uranyl acetate and lead citrate before being viewed under a Philips CM10 transmission electron microscope.
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RESULTS |
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Light microscopy.
Examination of the canine rectoanal region at the level of the light
microscope revealed heterogeneity in the thickness and organization of
the circular and longitudinal muscle layers. In the proximal rectum (8 cm from the anal verge) the circular and longitudinal muscle layers
were ~1.25 and 1.0 mm in thickness, respectively. The smooth muscle
cells within the rectum region were tightly packed together with few
septa separating the circular muscle layer into bundles (Fig.
1A). Closer to the IAS (4 cm
from the anal verge) discrete muscle bundles were apparent and were separated from each other by septae (Fig. 1B). At the level
of the IAS (0.5-1 cm from the anal verge), the circular muscle
layer was ~2.5 mm in thickness and was divided into circular muscle bundles, which were clearly separated by large connective
tissue. At the level of the IAS, the longitudinal muscle layer
was sparse and discontinuous. The circular muscle bundles varied
greatly in diameter from 100 µm or ~1/25th of the thickness of the
circular layer thickness to 350 µm or ~1/8th of the thickness and
were separated from each other by septae that also varied in thickness. Finer septae were also observed within individual muscle bundles and
these structures appeared to separate the larger muscle bundles into
smaller units that were ~20-30 µm in diameter (Fig.
1C).
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Immunohistochemistry.
To determine the distribution and shape of ICC networks within the
canine rectoanal region, we utilized immunohistochemistry by using
antibodies raised against the Kit receptor, which has been shown to be
highly specific for ICC in the GI tract (27). In the
proximal rectum, intense Kit-like immunoreactivity was observed in ICC
at the level of the submucosal surface of the circular muscle layer
(Fig. 2A). In this region, ICC
(IC-SM) lined this surface and were occasionally observed to follow the
contour of the septa, which divided the circular layer into separate
muscle bundles. Intense Kit-like immunopositive ICC were also located along the myenteric border, the interface between the circular and
longitudinal muscle layers. At this location, the myenteric ICC (IC-MY)
often formed a network that surrounded myenteric ganglia and were
located on both the circular and longitudinal muscle interfaces of
individual ganglia. In interganglionic regions, IC-MY formed a dense
network of cells, which had overlapping processes from adjacent ICC
(Fig. 2B). ICC were also observed within the circular muscle
layer (IC-IM) where the long axis of the IC-IM ran parallel to that of
the circular muscle fibers (Fig. 2B). Four centimeters from
the anal verge IC-SM were observed along the submucosal surface of the
circular layer, within the circular layer (IC-IM), and along the
myenteric border (IC-IM). In this region IC-IM had a spindle-shaped
morphology and ran on the outer aspects of circular muscle bundles that
were separated by septae (Fig. 2, C and D). IC-SM
and IC-MY also ran parallel to the circular muscle fibers but were
observed to run at different angles to this muscle layer (Fig.
2D).
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Electron microscopy. ICC throughout the canine rectoanal region possessed several ultrastructural features characteristic to all subpopulations of these cells. These features included a cytoplasm that was more electron dense compared with neighboring smooth muscle cells. ICC also possessed an abundance of mitochondria and a well-developed endoplasmic reticulum. Caveloae and a basal lamina were also associated with the plasma membranes of ICC and these structures distinguished these cells from fibroblasts or macrophages that were also occasionally observed in this region of the GI tract. Ultrastructural features, which distinguished these cells from neighboring smooth muscle cells, included a lack of thick filaments and few, if any, dense bodies.
ICC in the proximal rectum.
An ultrastructural examination of the submucosal surface of the
circular muscle layer in the canine proximal rectum revealed a dense
network of IC-SM (Fig. 3,
A-C). IC-SM
possessed many fine projections that extended off the main axis of the
cell in different directions (Fig. 3, A and B).
These processes came into close contact with processes of adjacent
IC-SM, where they often formed gap junctions (Fig. 3, D and
E, inset). The processes of IC-SM also extended
to neighboring circular smooth muscle cells with which they formed
close morphological associations, although definitive gap junctions
were not readily identified (Fig. 3A). IC-SM were also
observed to lie between enteric nerve bundles and smooth muscle fibers,
but close apposition between IC-SM and nerves were also not observed
(Fig. 3, B and D). Smooth muscle cells along the
submucosal surface of the canine rectum also formed gap junctional complexes with one another (Fig. 3F, inset).
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ICC in IAS.
As observed at the light microscopy level, the organization of the
circular muscle layer was very different in the IAS compared with the
proximal rectum. The circular muscle cells were separated into discrete
bundles by large connective tissue septae. Furthermore, the smooth
muscle cells near the submucosal surface were more irregular in profile
(Fig. 5A) compared with the
proximal rectum (Fig. 4A). We therefore sought to determine
whether the distribution of different classes of ICC was also different
in the IAS compared with the proximal rectum. ICC were observed at
several distinct locations in the IAS. An ultrastructural examination
of the submucosal surface of the circular muscle layer in the IAS
revealed a subpopulation of ICC (IC-SM) several cells deep (Fig.
5A). IC-SM were often observed adjacent to nerve bundles
(Fig. 5B) and formed gap junctions with the processes of
neighboring IC-SM (Fig. 5, C and D). The processes of IC-SM were also observed to form close association with
circular smooth muscle cells (Fig. 5E), although the
distinctive penta/septa laminate structures of gap junctions were not
observed. Gap junctions were also observed between circular smooth
muscle cells in the IAS, some of which had an atypical flattened
appearance with a highly irregular membrane profile (Fig.
5F, inset).
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DISCUSSION |
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In the present study, we examined the structural arrangement of the circular muscle layer and the accompanying distribution of specific populations of ICC within the canine rectoanal region. Functional studies of this region indicate there are significant differences in the motility patterns and electrical activity of the canine rectum and IAS (25). The present study indicates that the functional differences within this region are accompanied by differences in both the structural arrangement of smooth muscle cells as well as the distribution of ICC in this region of the GI tract.
From rectum to IAS, the time course and amplitude of pacemaker potentials change in an oral-to-aboral direction. In addition, a gradient in membrane potential exists across the thickness of the rectum with an apparent submucosal site of origin for slow-wave activity. In the distal direction, the electrical gradient becomes normalized across the circular muscle layer, and pacemaker potentials appear to emanate from throughout the muscle. (25). Our histological studies revealed that the circular muscle layer of the rectum consisted of a tight arrangement of muscle fibers occasionally separated by small connective tissue septae. The septae divided the circular layer into bundles. This structural arrangement is very similar to the circular muscle of the canine proximal colon (41). In the colon, slow waves arise from ICC along the submucosal surface of the circular muscle and then conduct across the muscle layer through low-resistance gap junction pathways that couple cells (2, 41). In contrast, the circular muscle layer in the IAS was arranged into discrete bundles separated from one another by large septae. Between the proximal rectum and IAS there was a increase in the number and size of septae that separated muscle bundles. Consequently, individual muscle bundles in the IAS constituted only a fraction of the entire width of the circular muscle layer, suggesting that regions of the IAS were functionally isolated from one another. A previous study (41) of the canine colon revealed that slow-wave activity was electrically coupled across septae. However, the colonic septae were much smaller than those found in the distal rectum or IAS. It would be difficult to perceive how electrical coupling could occur across such large extracellular spaces filled with connective tissue. The anatomical arrangement of the muscle in the IAS therefore supports our functional studies suggesting that electrical activity in the IAS emanates from numerous sites within the muscle layer, presumably from individual muscle bundles.
A large body of work now documents the important role that ICC play as generators of pacemaker potentials in the GI tract (15, 27, 39). A plexus of ICC has previously been described at both the submucosal and myenteric borders of the canine proximal and distal colons (2, 3, 40, 41), however, identification of these cells was based primarily on ultrastructural criteria, and it is difficult to visualize the extent and morphology of ICC networks by using electron microscopy alone. Methylene blue, rhodamine 123 and NADH-diaphorase histochemistry have been applied to investigate ICC networks in the canine colon; however, these labels have problems with specificity and label other cell types including enteric nerves (23, 32, 37, 42). More recently, it has been shown that ICC express the receptor tyrosine kinase, Kit (16, 24, 34, 38, 39) and that antibodies against this receptor are highly specific to label these cells throughout the GI tracts of a variety of species including mouse, rat, guinea-pig, dog, and human (4, 8, 12, 13, 18, 34, 35). Kit immunohistochemistry therefore represents an important additional methodology to visualize the overall distribution of ICC within the tunica muscularis. Kit immunoreactive ICC have previously been reported in the canine stomach (13), but there have been no studies on the canine large intestine or rectoanal region to date. Our Kit immunohistochemical observations of canine rectoanal muscles revealed that ICC were present 1) along the submucosal surface of the circular muscle layer; 2) at the level of the myenteric plexus, between the circular and longitudinal muscle layers; and 3) within the circular and longitudinal muscle layers. However, the relative densities of these cells varied from rectum to IAS.
Our immunohistochemical studies revealed that the rectum had distinct networks of ICC at the submucosal and myenteric extremes of the circular muscle layer. In contrast to the rectum the IAS possessed more diffuse Kit-labeling throughout the muscle layer. Immunoreactive ICC in the IAS possessed spindle shaped morphologies, whereas myenteric and IC-SM in the rectum were more stellate in appearance. The density of ICC in the IAS was not as great as that observed at either the submucosal or myenteric edges of the rectum. Within the IAS, ICC were generally localized to the periphery of muscle bundles or in septal structures. Thus we found that changes in the structural arrangement of circular muscle cells from rectum to IAS were accompanied by changes in the density and distribution of Kit-immunopositive ICC networks.
Distribution of Kit immunoreactive cells in the human rectoanal region is similar to what we observed in the canine model. ICC were identified in the human rectum within the longitudinal and circular muscle layers as well as at both plexus regions (10, 11), ICC were also identified within the IAS where they were reported to be infrequently scattered among smooth muscle fibers. The density of ICC in the IAS was also reported to be significantly lower than that observed in the circular muscle layer of the rectum. In these studies (10, 11) it was concluded that the distribution of ICC in this region of the GI tract was consistent with the proposed roles of ICC as rectoanal pacemakers, intermediaries of the neural control of muscle activity, and coordinators of muscle activity.
In the human small intestine discrepancy exists as to whether all subpopulations of ICC are labeled with Kit antibodies (26, 33). This could be attributable to different antibodies to the Kit receptor that have been used in these studies. To ensure that we were able to label all subpopulations of ICC and to obtain a more complete appraisal of the structure of ICC in the rectoanal region we supplemented our Kit-immunohistochemical identification of ICC with ultrastructural studies by using electron microscopy. With the use of this approach, we found that ICC in the canine rectoanal region possessed a set of cellular features that, when taken together, distinguished these cells from smooth muscle cells or from other cells, such as fibroblasts, macrophages, mast cells, or nerve elements also found in the tunica muscularis of the canine GI tract. These features included: 1) an electron-dense cytoplasm, 2) an abundance of mitochondria in the cytoplasm along with prominent rough endoplasmic reticulum compared with neighboring smooth muscle cells, 3) numerous caveolae in the plasma membrane along with a continuous basal lamina, 4) a lack of thick filaments and few if any dense bodies within the cytoplasm, and 5) numerous fine projections that extended off the main axis of the cell that came into close contact with adjacent ICC and in some instances with smooth muscle cells, where they often formed gap junctions. ICC were also associated with enteric nerves, although synapse-like membrane densifications like those seen in the murine and canine stomachs and in the murine proximal colon (1, 13, 36) were not evident.
Arrangement of ICC in the rectoanal region changed as the number and size of septae increased in the oral to aboral direction. In the canine rectum, ICC identified by using ultrastructural criteria were primarily located along the submucosal surface of the circular muscle layer and at the level of the myenteric plexus. Although there was a dense network of IC-SM along the submucosal surface and these cells formed gap-junctions with one another, distinct gap junctions were not readily identified between IC-SM and smooth muscle. This could be a consequence of fixation or a difference in the number or size of junctions that exist between IC-SM and smooth muscle cells. Previous studies of the canine proximal colon have suggested that IC-SM express specific ionic conductances that lead to greater polarization of cells in this region and consequently to a electrical gradient in membrane potential from submucosal to myenteric borders (29, 41). Indeed, we observed a similar gradient in membrane potential across the thickness of the circular muscle layer of the rectum (25). The normalization of the electrical gradient in the anal direction may be the consequence of decreased numbers of IC-SM. In the IAS, ICC were distributed throughout the muscle thickness and were located at the interface between smooth muscle bundles and septa. The processes of ICC at this interface overlapped, forming a network around individual muscle bundles. The density of ICC identified by using electron microscopy was at least as great as that seen by using Kit immunohistochemistry. The presence of ICC throughout the thickness of the IAS and the absence of a distinct plexus region at either border once again supports the hypothesis that pacemaker potentials in this region are generated from throughout the muscle thickness (25).
In summary, the transformation in the structure of the circular muscle layer and changes in the distributions of ICC in the terminal regions of the canine GI tract could explain the differences in the electrical activity recorded in the proximal rectum vs. that of the IAS.
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ACKNOWLEDGEMENTS |
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The authors are extremely grateful to Julia R. Bayguinov for excellent technical assistance with the immunohistochemical studies.
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FOOTNOTES |
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This work was supported by National Institute of Diabetes and Digestive and Kidney Diseases Grants DK-40569 and DK-57236. The Morphology Core Laboratory was supported by Program Project National Institute of Diabetes and Digestive and Kidney Diseases Grant DK-41315, which was used for both immunohistochemical and electron microscopy studies.
Address for reprint requests and other correspondence: S. M. Ward, Dept. of Physiology and Cell Biology, Univ. of Nevada School of Medicine, Reno, NV 89557 (E-mail: sean{at}physio.unr.edu).
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
First published January 22, 2003;10.1152/ajpgi.00294.2002
Received 22 July 2002; accepted in final form 2 December 2002.
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