Distribution of interstitial cells of Cajal in tunica muscularis of the canine rectoanal region

Kazuhide Horiguchi, Kathleen D. Keef, and Sean M. Ward

Department of Physiology and Cell Biology, University of Nevada School of Medicine, Reno, Nevada 89557


    ABSTRACT
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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


    INTRODUCTION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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.


    MATERIALS AND METHODS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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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.


    RESULTS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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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Fig. 1.   Gross morphology of the tunica muscularis within the canine rectoanal region. A: transverse section through the circular muscle (CM) layer 8 cm proximal to the anal verge. CM fibers in this region were tightly packed with occasional thin septae (arrow). B: transverse section taken from the same animal at a distance 4 cm proximal to the anal verge. In this region, the CM is less tightly packed and muscle bundles were separated by septae (arrows). C: at the level of the internal anal sphincter (IAS) the circular layer was divided into discrete muscle bundles by large septae (arrows). Smaller septae can also be seen within individual muscle bundles (arrow heads). LM, longitudinal layers; SMB, submucosal border; MB, myenteric border.

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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Fig. 2.   Kit-receptor immunohistochemistry revealed the distribution of interstitial cells of Cajal (ICC) in the rectoanal region. A and B: ICC in the rectum (8 cm from the anal verge). C and D: ICC taken 4 cm from the anal verge. E and F: ICC within the IAS (1 cm from the anal verge). A: Kit-like immunopositive ICC were observed along the surface of the SMB of the CM layer in the proximal rectum. Submucosal ICC (IC-SM) formed an intensely labeled layer along the SMB (*) and also along septae that divided the CM into bundles. Intramuscular ICC (IC-IM) within the circular layer (CM) were also visible (arrows). B: Kit-like immunopositive ICC at the level of the myenteric (MY) plexus (IC-MY, arrow heads) and within the circular layer (IC-IM, arrows) of the rectum. IC-MY formed a dense network of cells located on the circular and longitudinal aspects of MY ganglia and within interganglionic regions. IC-IM were spindle shaped with their main axis running parallel to the CM fibers. C and D: distribution of ICC, 4 cm from the anal verge. ICC were located along the SMB (*), within the CM layer (arrows) and along the MY border (arrow heads). D: IC-IM were spindle shaped in morphology and were located on the outer aspects of CM bundles. E and F: ICC within the SM surface of the CM and in the bulk CM of the IAS. ICC (arrows) in the IAS possessed a spindle-shaped morphology and were located on the outer aspects of CM bundles. Scale bars in B, D, and F applies to A and B, C and D, and E and F, respectively.

At the level of the IAS, there was no obvious myenteric or submucosal network of ICC like that observed in the rectum. In this region, the ICC possessed a spindle-shaped morphology, were predominately orientated in the same direction as circular smooth muscle fibers, and were usually located on the surface of septae that separated the circular muscle into discrete bundles (Fig. 2, E and F).

To confirm the distribution and examine the ultrastructural features of different classes of ICC and their relationships to other cell types in the canine rectoanal region, we performed electron microscopy on the tunica muscularis of the proximal rectum and IAS.

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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Fig. 3.   Ultrastructure of the SM surface of the CM layer in the proximal rectum (8 cm from the anal verge). A: IC-SM (asterisks) along the SM interface of the CM layer. IC-SM possess an electron-dense cytoplasm and were closely associated with smooth muscle cells (arrows, ×7,000 magnification). B: processes of IC-SM (*) intervened between nerve bundles (N) and adjacent smooth muscle cells (×5,600 magnification). C: at higher magnification the cytoplasmic features of IC-SM can be seen. Numerous mitochondria, rough endoplasmic reticulum, caveolae, and a basal lamina are typical of IC-SM (×15,200 magnification). D: close contact between adjacent IC-SM (arrow) is shown (×13,700 magnification). E: a higher power image of the close contact (putative gap junction) indicated by the arrow in D is shown (×80,000 magnification). Inset, gap junction between the processes of IC-SM (×72,000 magnification). F: innermost circular smooth muscle cells formed gap junction with each other (arrow; ×23,600 magnification). Inset, higher magnification (×78,000) of the gap junction indicated by the arrow in F.

Under the electron microscope, the bulk circular muscle of the proximal rectum appeared tightly packed in transverse section with occasional fine septae separating muscle cells into poorly defined bundles (Fig. 4A). Occasional IC-IM were observed interposed between muscle fibers in this region of the GI tract along with nerve bundles and blood vessels (not shown). ICC located at the interface of septae and the circular smooth muscle bundles were more abundant than IC-IM. These cells overlapped one another, forming a network that was often several cells thick, and their processes came into close association with one another. IC-SEP populated septae throughout the circular muscle layer and occasionally septae and IC-SEP spanned the entire distance from the submucosal to the myenteric surface. IC-SEP were also closely apposed to nerve bundles were observed in septal structures (Fig. 4B).


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Fig. 4.   Ultrastructure of bulk CM, septa, and MY regions in the proximal rectum (8 cm from the anal verge). A: typical morphological arrangement of bulk CM cells at low magnification. Occasional ICC were observed within the muscle bundles, but none are apparent. Thin septal structures can be seen to divide the tightly packed CM fibers (arrows; ×2,500 magnification). S, Schwann cell. B: ICC located within septa (IC-SEP) (*) that separated the CM into bundles. IC-SEP were observed at the interface between septal structures and CM bundles. Nerve bundles were often observed adjacent to IC-SEP (×4,800 magnification). The cytoplasm of IC-SEP was electron dense compared with the circular smooth muscle cells and contained numerous mitochondria (B, inset). The plasma membrane had numerous caveolae and a basal lamina associated with it (×8,900 magnification). C: ICC at the level of the IC-MY. IC-MY (*) also possessed an electron-dense cytoplasm and numerous mitochondria. Caveolae were also observed in the plasma membrane (×3,800 magnification). IC-MY possessed numerous processes that extended from the perinuclear region of the cell and these came into contact with adjacent IC-MY to form gap junctions (inset; ×112,000 magnification). IC-MY were observed on both the circular and longitudinal aspects of MY ganglia. D: IC-MY are also located on the longitudinal side of MY ganglia (G; ×5,300 magnification).

At the level of the myenteric plexus, ICC (IC-MY) were also observed at the interface between the circular and longitudinal muscle layers (Fig. 4C). IC-MY in this region possessed many of the characteristic features of ICC in other regions of the GI tract of several animal species (18), in that they possessed numerous thin processes that extended from the main body of the cell in many directions and came into close association with adjacent IC-MY to form gap junctions (Fig. 4C, inset). In interganglionic regions, IC-MY were observed as a thin network of cells, but in regions in which myenteric ganglia separated the circular and longitudinal muscle layers, IC-MY were observed on both the circular and longitudinal aspects of myenteric ganglia (Fig. 4D).

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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Fig. 5.   Ultrastructure of the SM surface of the CM layer in the IAS. A: image of the SM surface of the CM layer taken at low magnification. IC-SM (*) and nerve bundles were located in this region (×3,600 magnification). B: higher power image of A. IC-SM possessed an electron dense cytoplasm, many mitochondria, rough endoplasmic reticulum, and caveolae (×11,400 magnification). C: IC-SM formed gap junctions (arrow) with neighboring IC-SM (×20,400). D: higher magnification of the gap junction indicated by the arrow in C (×91,000 magnification). E: processes of IC-SM were closely associated with each other (arrows) and with smooth muscle cells (double-headed arrows; ×18,100 magnification). Gap junctions were observed between the innermost CM cells (F; arrows; ×9,900 magnification). The gap junction between the inner circular smooth muscle cells shown by the arrow in F is shown at higher magnification in the inset (×96,000 magnification).

Because the circular muscle layer of the IAS was divided into small bundles of muscle fibers separated by large septae, we considered the possibility that IC-SM were a subpopulation of ICC located on the outer aspects of circular muscle bundles (IC-SEP). IC-SEP were observed at the interface between septal structures and circular muscle bundles (Fig. 6A). IC-SEP had a slender profile with the main axis of the cell running parallel to the circular muscle fibers. IC-SEP networks were several cells deep (Fig. 6, B and C), and the processes of these cells formed an overlapping network (Fig. 6, C-E). The processes of IC-SEP formed numerous gap junctions with one another (Fig. 6, D-F) and with neighboring circular smooth muscle cells (Fig. 6F). The ultrastructure of IC-SEP was similar in detail to IC-SM, and they also possessed all of the characteristics common to ICC (Fig. 6C).


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Fig. 6.   Ultrastructure of IC-SEP in the canine IAS. A: IC-SEP (*) were located at the interface between a septal structure and CM bundles. IC-SEP were found adjacent to smooth muscle cells in septae (×3,800 magnification). B: IC-SEP and their processes (*) ran adjacent to CM bundles within septa. Nerve bundles were often associated with these cells (×4,400 magnification). C: cytoplasm of IC-SEP in the neighboring section of B is shown at higher magnification. IC-SEP possessed an electron-dense cytoplasm, numerous mitochondria, and an abundance of rough endoplasmic reticulum. Caveolae and basal lamina were also observed along the plasma membranes of IC-SEP (×15,500 magnification). D: processes of IC-SEP (*) formed multiple gap junctions with each other (arrows) (×13,500, magnification). E: higher power image of D (×30,200 magnification). The gap junctions indicated by the arrow are shown in the inset (×73,000 magnification). F: IC-SEP also formed gap junctions with neighboring circular smooth muscle cells (arrow). A gap junction between two IC-SEP is also seen (double-headed arrow; ×19,000 magnification).

The structural arrangement of the circular muscle layer of the IAS observed by using light microscopy was more apparent at the level of the electron microscope. In cross section, bundles of bulk circular muscle fibers were separated by large septae, such that some small bundles only had 15-20 smooth muscle cells grouped together (Fig. 7, A and B). The circular muscle fibers within bundles had a typical smooth muscle morphology and formed gap junctions with adjacent smooth muscle cells in the same bundle (Fig. 7C, inset). Septa had numerous collagen fibrils, nerve bundles, blood vessels, fibroblasts, mast cells, and ICC associated with them and varied in thickness throughout the circular layer (Fig. 7, A and B). Nerve bundles in septae were occasionally varicose in nature, containing large, dense, cored, and small clear vesicles (Fig. 7D). Occasional mast cells within septae contained numerous secretory granules and were observed adjacent to nerve bundles (Fig. 7E). Although the longitudinal muscle layer becomes diffuse in the region of the IAS (see Fig. 1 and Fig. 1 of Ref. 25), some longitudinal muscle was present. A similar structural arrangement to that found in the circular layer was also observed in the longitudinal muscle. Septa, which contained nerve bundles, blood vessels, and fibroblasts divided the longitudinal layer into discrete muscle bundles. Although nerve bundles were observed in the longitudinal muscle in the IAS, no ICC were observed. (Fig. 7F).


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Fig. 7.   Ultrastructure of the bulk muscle layers in the IAS. A and B: arrangement of smooth muscle cells in the bulk of the CM layer. No ICC were observed within the CM bundles or around smaller circular bundles. Large nerve bundles and a fibroblast-like cell (F) were also seen within the septa (×2,000 magnification). C: circular smooth muscle cells shown at higher magnification revealing gap junctions between one another (arrow; ×10,500 magnification). The gap junction indicated by the arrow is shown at higher power in the inset in C (×62,000 magnification). D: A nerve bundle running within the CM bundle. Varicose regions of axons within nerve bundles contained electron dense cored vesicles. No ICC were seen in association with these nerve bundles (×14,500 magnification). E: a mast cell within the CM layer that contained many secretory granules is shown (×8,000 magnification). F: nerve bundles running within the longitudinal muscle layer are shown. ICC were not observed in close morphological association with nerve bundles at this level in the IAS (×4,800 magnification).

ICC were also observed at the level of the myenteric plexus in the IAS. In this region of the IAS, IC-MY were characterized by possessing a thin profile with numerous slender cellular processes extended from the main body of the cell, and these interdigitated with processes from adjacent IC-MY (Fig. 8, A-C). Within the cytoplasm of IC-MY there was an abundance of rough endoplasmic reticulum, which often appeared to surround the numerous mitochondria in these cells. Nerve bundles that contained large dense-cored and small, clear vesicles were observed in this region adjacent to IC-MY (Fig. 8B). At higher magnification, the slender processes of IC-MY formed gap junction complexes with processes of neighboring IC-MY (Fig. 8D, inset).


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Fig. 8.   Ultrastructure of IC-MY in the canine IAS. A: low power image revealing the processes of electron dense IC-MY (*) adjacent to the CM layer. Nerve bundles were observed in this region adjacent to IC-MY (×5,200 magnification). B: processes of IC-MY (asterisk) were partially surrounded by circular smooth muscle cells (×4,600 magnification). C: cytoplasm of IC-MY is shown at higher magnification. Numerous mitochondria, a well-developed rough endoplasmic reticulum, and caveolae (arrowheads) were readily observed within IC-MY (×16,200 magnification). D and inset: IC-MY formed gap junctions with each other (arrow; ×16,600 magnification). The gap junction indicated by the arrow in D is shown at high magnification in the inset (×80,000 magnification).

A diagrammatic representation of the distribution of ICC in the circular muscle layer of the canine rectum and in the IAS is illustrated in Fig. 9.


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Fig. 9.   Diagrammatic representation of the distribution of ICC in the CM layer of the canine rectoanal region. A: distribution of ICC in the rectum (8 cm from the anal verge; black cells). ICC were distributed along the SM and MY borders and within the circular layer. B: diagrammatic representation of the distribution of ICC in IAS (1 cm from the anal verge). ICC were more diffusely distributed throughout the CM layer, primarily at the interface between muscle bundles and septae.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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.


    ACKNOWLEDGEMENTS

The authors are extremely grateful to Julia R. Bayguinov for excellent technical assistance with the immunohistochemical studies.


    FOOTNOTES

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.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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