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Interstitial cells of cajal in human small intestine Ultrastructural identification and organization between the main smooth muscle layers
GASTROENTEROLOGY
1991;100:1417-1431
Interstitial Cells of Cajal in Human Small Intestine Ultrastructural Identification and Organization Between the Main Smooth Muscle Layers JCRI JOHANNES Anatomy Department
RUMESSEN C, University
and LARS THUNEBERG
of Copenhagen,
Previous morphological and electrophysiological studies have supported the hypothesis that interstitial cells of Cajal have important regulatory (pacemaker) functions in the gut. In the current study, interstitial cells of Cajal associated with Auerbach’s plexus in human small intestine were studied. Freshly resected intestine was examined by light and electron microscopy. The interstitial cells of Cajal resembled modified smooth muscle cells. They had caveolae and dense bodies, an incomplete basal lamina, a very well-developed smooth endoplasmic reticulum, and abundant intermediate (10nm) filaments. Myosin filaments were not seen. Fibroblastlike cells were distinguished by their lack of caveolae and dense bodies, the relative scarcity of smooth cisternae and intermediate filaments, and the abundant granular endoplasmic reticulum. Interstitial cells of Cajal were arranged in networks of bundles containing processes of two to seven cells with fibroblastlike cells interspersed in the bundles. The bundles were innervated by nerve elements of Auerbath’s plexus and extended into both layers of smooth muscle, between muscle cells, and into septa. The bundles were closely associated with elastin fibers. The organization shown in this study strongly supports the concept of interstitial cells of Cajal as important regulatory cells also in the human small intestine. The characteristic cytology and organization of interstitial cells of Cajal may provide a basis for future morphological, electrophysiological, and pathological studies of these cells in human small intestine.
0
ur
previous studies of the structural organization of interstitial cells of Cajal (ICC) located in the small intestine of the mouse led to the proposal that these cells had important regulatory functions and that a subpopulation of ICC located between the main
Panum Instituttet,
Copenhagen,
Denmark
muscle layers were ideal candidates for small intestinal pacemaker cells (l-5). Morphological and electrophysiological studies of several species including humans have supported this hypothesis (6-8). However, evidence of ICC in the human small intestine is scanty (9). Increasing attention is being given to motility disorders of the small intestine (10). Detailed knowledge of the structural and physiological characteristics of regulatory and slow wave-generating cell populations is therefore of fundamental importance and may give new insights into the control of gastrointestinal (GI) motility and changes in disease. We have therefore studied the ultrastructure of ICC located between the main muscle layers of the human small intestine. Materials
and Methods
Freshly resected, uninvolved small intestine (two duodenum, two jejunum, and seven terminal ileum) was obtained from nine adult patients (three female and six male; age, 32-90 years; median, 64 years) undergoing surgery because of incarceration of inguinal hernia or because of gastric, pancreatic, or colonic carcinoma. The patients had no other GI disease. They were not taking any drugs known to affect the structure of the enteric nerves or musculature. Patients with peritoneal carcinosis were excluded. After resection, l-Z-cm pieces of small intestine were immediately cut and immersed in two types of fixatives: (a) modified Karnovsky’s fixative (11) containing either 2% + 2%, 8% + 8%, or 12% + 12% glutaraldehyde (Taab, Berkshire, United Kingdom) and paraformaldehyde (Merck,
EM, electron microscopy; cells; gER, granular endoplasmic reticulum; ICC, interstitial cells of Cajal; sER, smooth endoplasmic reticulum; SMC, smooth muscle cells. o 1991 by the American Gastroenterological Association 0016-5085/91/$3.00 Abbreviations
FLC, fibroblastlike
used in this paper:
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sections were cut, mounted on copper grids, and contrasted with uranyl acetate and lead citrate. The grids were examined in Philips EM 300 and 400 electron microscopes (Eindhoven, The Netherlands). The study was performed in accordance with the Helsinki Declaration II and approved by the ethical committees of Copenhagen and Copenhagen County.
Results The muscle layers were thicker in the proximal small intestine, but the overall organization was similar in different regions (duodenum, jejunum, and ileum). Illustrations are mainly from the ileum, which was studied most extensively. Light Microscopy
Figure 1. Longitudinal section of the musculature in ileum. The circular layer (CM) consists of parallel laminae separated by connective tissue septa in continuity with the submucosa (SU). SE, serosa; LM, longitudinal muscle: long orrow, ganglion of Auerbach’s plexus: short arrow, subserous vessel with erythrocytes; arrowheads, innermost circular layer (toluidine blue; original magnification X 100; bar = 100 pm).
Darmstadt, Germany) in 0.1 mol/L phosphate buffer (Merck), pH 7.3, at 20°C for at least 2 hours; and (b) modified Bouin’s fixative containing 2% + 2% or 4% + 4% glutaraldehyde and paraformaldehyde in 0.1 mol/L phosphate buffer with picric acid (Merck) added from a saturated solution to give a final concentration of 0.1896, pH 7.3, at 20°C for at least 2 hours. After immersion, 1 x 2-mm pieces were cut and left in fixative overnight, rinsed for 60 minutes in 0.1 mol/L phosphate buffer, pH 7.3, and postfixed in 2% 0~0, in 0.1 mol/L phosphate buffer for 2 hours. The pieces were dehydrated in alcohol, block stained with alcoholic uranyl acetate, and embedded in Epon 812 R (Merck). Thin sections were stained with toluidine blue for light microscopical investigation, Suitable areas were selected for transmission electron microscopy [EM), and ultrathin (70 nm)
The general plan of the small intestinal muscularis externa is shown in Figure 1. The longitudinal muscle layer had a total thickness averaging 50-100 cells. The layer was subdivided by irregular connective tissue septa containing the larger nerves and vessels (Figure 1). Muscle cells with a similar orientation often surrounded ganglia and primary fascicles of the myenteric plexus, which were therefore partly or totally embedded in the longitudinal muscle layer (Figure 2) but never in the circular layer. In ganglionated areas, a large connective tissue space divided the main muscle layers, whereas in other areas the muscle layers were in close contact (Figure 2). The space contained smooth muscle cells (SMC), interstitial cells, vessels, and nerve elements of Auerbach’s plexus. Interstitial cell types of slightly different staining intensity were seen between the muscle layers, but specific cell types could not be identified with certainty (Figure 3A).
Figure 2. Ganglion (G) in Auerbach’s plexus surrounded by longitudinal muscle (Lhf), merging (arrows) with the circular layer (CM) (toluidine blue; original magnification x 240; bar = 50 nm).
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Figure 3. A. Septum (S) in longitudinal muscle layer (LM).Arrows, secondary nerve strands of Auerbach’s plexus; CM, circular muscle; upper frame, Figure 3C; lowerframe, Figure 3B; arrowheads, ICC processes (toluidine blue, original magnification x 800; bar = 10 pm). B. Lower frame in Figure 3A. Nucleus (N) and processes (*) of ICC located between the main muscle layers at the level of Auerbach’s plexus. Smooth cisternae are prominent in the processes (arrowheads). NF, tertiary nerve fascicles of Auerbach’s plexus (original magnification x 7500; bar = 2 km). C. Upper frame in Figure 3A. Processes (*) of ICC extending into septum between longitudinal muscle cells (LM). Processes of ICC are in intimate contact with two FLC (nucleus of the lower is indicated by N). M, SMC; NF, nerve fascicle (original magnification x 7500; bar = I km).
The circular muscle layer was divided by connective tissue septa in circularly oriented (ring-shaped) lamellae with a width of 50-150 pm running along the whole circumference (Figure 1). The septa were in continuity with the submucous layer and extended through the full thickness of the circular layer. A subdivision of the circular lamellae consisting of the innermost one to five cells was distinguishable in
most well-oriented sections, separated from the outer layer by a connective tissue space containing nerve elements of the deep muscular plexus, interstitial cells, and vessels. The innermost subdivision of the circular layer could be distinguished as covering the inner one fourth to one third of the muscular lamellae, after which it seemed to merge with the bulk of the circular lamellae.
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Smooth Muscle Cells
Figure 4. Longitudinally sectioned SMC (M) and ICC process (*). Interstitial Cells of Cajal cytoplasm contains irregularly arranged intermediate filaments (IF), and a few dense bodies are seen (arrowheads). Myosin filaments and basal lamina are absent in contrast to SMC, in which myosin is depicted by arrowheads and dense bodies by LUTOWS(original magnification ~15,000; bar =
In low-power EM, SMC were often impossible to distinguish from ICC (see later). At higher magnification, SMC of both layers were easily distinguished by their size, arrangement, and in particular their characteristic arrangement of dense bodies and conspicuous myosin filaments (Figure 4). Caveolae were frequent, arranged in longitudinal bands alternating with dense bands (Figures 5B and 6). Cytoplasmic dense bodies were very conspicuous and regularly distributed (Figure 4). Submembraneous smooth endoplasmic reticulum (sER) and rows of mitochondria were characteristically located close to the caveolated membrane areas. Other organelles were concentrated in the larger perinuclear regions. The nucleus had sparse, peripherally distributed het,erochromatin (Figure 5A). The SMC surrounding the ganglia and larger fascicles of Auerbach’s plexus had the same ultrastructural features. Muscle cells in the circular layer were interconnected by adherens-type and gap junctions. The latter were not seen in the longitudinal layer. No mitoses were seen in SMC. Nerve terminals were only occasionally in close, synapselike contact with SMC of either layer (Figure 6). The characteristic features are summarized in Table 1. Interstitial Cells of Cajal
1 w).
Iden tifca tion The outer, main layer of circular muscle was about twice the thickness of the longitudinal muscle layer and generally comprised 150-200 cells in the ileum. Both the longitudinal and circular layers were pierced by smaller connective tissue septa containing capillaries and tertiary nerve elements accompanied by interstitial cell types. The larger connecting nerve strands and vessels ran in the septa between the circular muscle lamellae.
The problem of identifying ICC in sections is best illustrated by considering Figures 5A, 7A, and 8. In these low-power electron micrographs ICC may be difficult to distinguish from fibroblastlike cells (FLC) and especially from SMC. The distinctive cytological features are shown at higher magnifications (Figures 5B and C and 7B and C), in which the abundance of intermediate filaments and sER is most characteristic. Caveolae, dense bodies, and basal lamina are variably developed.
Electron Microscopy The ultrastructural preservation of the superficial parts of the musculature (from serosa to the outer parts of the circular muscle) was generally satisfactory and allowed detailed cytological studies with all fixatives used. Axonal varicosities and neuronal mitochondria were often suboptimally preserved, especially in the deeper layers of the circular muscle, whereas muscle and interstitial cells were well preserved at corresponding levels. The tissue in the deep layers was better preserved when the fixative with the highest aldehyde concentrations was used. Glycogen granules were most prominent in the modified Bouin’sfixed material.
Cytological Features Light microscopy. In toluidine blue-stained sections, cells with abundant, lightly stained cytoplasm and an ovoid nucleus with a finely granular chromatin were visible (Figure 3A). The nuclear features did not suffice to distinguish ICC from other interstitial elements, and their processes were very similar .to SMC processes (Figure 3A). Electron microscopy. Profiles of ICC processes were long with only little apparent branching (Figure 5A). Processes of ICC were narrower and of more heterogeneous size than those of SMC (Figures 4 and 5A). Organelles seemed heterogeneously distributed
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region in ileum. Interstitial cells of Cajal processes (*) partially Figm re 5. A. Survey of the intermuscular form bundles extending into the longitudinal muscle layer (LM). Frames b and c are magnified in Figure x 2800; bar = 10 km). (Figure continues on page 1422.) cell (original magnification
in ICC processes, which were dominated by a very well-developed sER in broad peripheral zones (Figures 3B and 5B) and a dense network of intermediate (10 nm) filaments (Figures 4 and 7B). Intermediate filaments were organized in bundles, more parallel in the smaller processes. Other (few) ICC processes were dominated by large mitochondria with flattened cristae (Figure 7C). Microtubules were often seen; granular endoplasmic reticulum (gER) was inconspicuous. Glycogen granules were most often evenly distributed within processes, and they had a density and size comparable to those in SMC. Some ICC processes were packed with clumps of glycogen (Figure 9). The perinuclear regions of ICC (Figures 8 and 10) had abundant cytoplasm with a crowding of gER and Golgi stacks but contained less sER than ICC pro-
CELLS OF CA)AL
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ensheath nerve elements (NF] I and 4B and C. F, FLC; End, endotb elial
cesses. Intermediate filaments were also abundant in perinuclear regions (Figure 10). The ICC nuclei (Figures 3B and 10) had a chromatin pattern similar to SMC nuclei, and the contours were often rather irregular. The ICC had a variably developed basal lamina, most prominent in regions of dense bands and in some ICC located inside the muscle layers (Figures 7C and 11). Caveolae were distinctive, of similar appearance as in SMC, but clearly fewer in number (Figures 10 and 11). Caveolae alternated only at times with dense bands (Figure 11); a clear distinctive feature, as opposed to SMC, was the relatively few dense bands (Figures 4 and 5B). The ICC cytoplasm contained conspicuous dense bodies, although smaller and more irregularly distributed than in SMC (Figures 11 and 12).
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Figure 5 (cont’d.). B. Bundle of 5 (l-5)ICCprocesses dominated by sER (arrowheads) and intermediate filaments. NF, tertiary nerve elements; M, SMC in longitudinal layer; End, endothelial cell (original magnification X 12,000; bar = 1 pm). C. Cross-sectioned ICC bundle (*) and a single SMC (M). At the bottom a pericyte (P) and an endothelial cell (End) are seen. NF, tertiary nerve fascicle; N, nucleus of ICC (original magnification X8500; bar = 2 km).
Thick (15 nm, myosin) filaments were not seen in ICC in sharp contrast to the abundance of myosin filaments in SMC (Figure 4). Thin filaments (5 nm, actin) were rare, mostly located in peripheral zones of ICC cytoplasm. Another characteristic (although not specific) feature of ICC was the occurrence of lipid droplets (Figure 10). These droplets were either homogeneous and moderately electron dense, or they were more heterogeneous. A unit membrane could not be dis-
cerned. Occasionally a single cilium was seen. Coated vesicles and free ribosomes were frequent [Figures 10 and 12C). The cytology of ICC compared with other cell types in the same area is summarized in Table 1. Organization Processes of ICC were very abundant in ganglionated areas, distributed all over the intermuscular
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plane within the dense collagen mat (Figure 5A). The ICC formed incomplete sheaths around ganglia and smaller fascicles and were mostly arranged in groups and bundles of three to seven cells with large areas of membrane closely apposed and separated by a rather constant cleft of about 20 nm (Figure 5B). The density and size of ICC bundles seemed greatest in the duodenum. Adherens-type junctions between ICC were seen, but gap junctions were not encountered. The ICC bundles extended from the intermuscular plane into the longitudinal layer (Figures 3C and 5A) or into the circular layer (Figure 7). The ICC penetrated both layers into the main septa (Figures 3 and 7) as well as in between SMC (Figure 5A). Gap junctions between ICC and other cells including SMC were not seen. The ICC bundles were always in intimate contact with nerve elements of Auerbach’s plexus, and synaptic specializations with clustering of vesicles and presynaptic densities were frequent (Figures 8 and 13). These varicosities contained either small 50-nm agranular vesicles together with 100-nm granular vesicles or 50-nm agranular vesicles together with large (150-ZOO-nm) dense core vesicles. The ICC were often in intimate contact with FLC, and adherenstype (intermediate) junctions were occasionally seen (Figures 3C and 6). Fibroblastlike cells were interspersed in the ICC bundles and seemed to represent an extension of some of these in the deeper parts of both muscle layers (Figure 6). Close relations to macrophagelike cells were also seen but infrequently. The extracellular elastin had a typical and rather consistent localization and relation to ICC bundles (Figures 14 and 15). Elastin material in different stages of elastogenesis (fibrils and small or larger homogeneous fibers) were nearly always scattered around individual ICC, often in close contact with the plasma membrane (Figure 15). Elastin was located between individual ICC and between ICC and FLC or SMC (Figure 14). This arrangement suggested a more specific relation to ICC bundles and processes. Elastic fiber microfibrils were 10-15 nm long and had a distinct periodicity (Figures 12C and 15).
Nerve Elements
Figure 6. Connections between ICC (*) and FLC (short arrows) as well as FLC-SMC (long arrows) in the longitudinal muscle layer (M). Arrowheads, axon terminal in synapselike contact with SMC; NF, nerve fascicle; E, elastin (original magnification X 10,000; bar = 2 pm).
Ganglia and primary fascicles of Auerbach’s plexus were closely apposed to or embedded in the longitudinal muscle layer (Figures 1 and 2). Secondary and tertiary elements ramified in both muscle layers as well as in the intermuscular plane and connective tissue septa. Tertiary elements (3-15 axons) followed processes of ICC closely. Three main types of nerve varicosities were distinguished based on their content of vesicles (Figures 8 and 13): (a) 50-nm small agranular vesicles together with 100-nm
Table 1. Distinctive Cytological Features of Different Cell Types Associated With Auerbach’s in the Human Small Intestine. Cell type ICC cell bodies ICC processes SMC FLC MLC Glial cell Endothelial cell
Ca
Bm
sER
gER
++ ++ +++ 0 0 0 +++
+ + +++ 0 0 +++ +++
++ +++ ++ + + + +
(1, + +++ + + +
DB
M
I
A
Li
+
+
0
++
0 0
++ +++
+ +
++ ++
+ ++ +++ + +
+++ 0 0 0 0
+++ 0 0 0 0
Mi
cv
LY
++
++
+ ++ ++ + + +
(1, + +++ 0 0
Plexus
++ + + ++ +
+++
+
(+I (+I 0 0
+ 0 + 0
NOTE. An approximation of relative frequency is indicated by pluses (+-+ + +); 0 or (+) means not encountered or inconspicuous. Ca, caveola; Bm, basal lamina; Mi, mitochondria; Cv, coated vesicles; Ly, lysosomes; DB, dense bands/bodies; M, thick filaments; intermediate filament: A, thin filaments: Li, lipid droplets.
I,
Figure 7. A. Interstitial cells of Cajal processes (*) running in a septum between circular muscle cells (CM]. Gc, glial cell with nerve profiles; frames, detailed in B and C; IM, intermuscular plane (original magnification x 6000; bar = z urn). B. Upper frame in A, neighboring section. The ICC cytoplasm is dominated by intermediate filaments (IF). Broad arrow, FLC process; arrowheatl, dense band (original magnification ~13,000; bar =
1 km). C. Lower frame in A, neighboring section. This ICC is dominated by numerous large mitochondria (Mi) and a continuous basal lamina is apparent (arrowheads). M, muscle cells (original magnification ~14,000; bar = 1 km).
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Figure 8. Organization of ICC processes (*) and smaller nerves (NF) between a ganglion (G) and the longitudinal muscle 1~ iyer (L14. 1nterstiti: 11cells of Cajal processes are in close contact with a tertiary nerve fascicle (NF) with a naked terminal containing lauge der we core vf !sic:les (long arrow). Short arrows, process of an FLC; M, SMC; E, elastin (original magnification x 12,500; bar = 1 pm).
Figure 9. Interstitial cells of Cajal processes (*) as they appear after fixation with a fixative containing picrate (modified Bo uin’ Glycog en granules are well preserved (arrows). The heterogeneous distribution within and in between different processes is aplparent. E, elastin fibIrils; * , ICC processes; C, collagen fibrils (original magnification x 22,500; bar = 1 urn).
a.
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Figure 10. P&nuclear region of ICC showing dense loops and bundles of intermediate filaments (IF) with regional clusteri ng of mitoch .ondria (Mi) and gER.Caveolae are indicated (arrowheads). M, SMC; N, ICC nucleus; arrow, lipid droplets with flocr :ulent al. *Process of another ICC (original magnification X 14,000; bar = 1 km).
large dense core vesicles; (b) 50-nm small agranular vesicles together with 150~200-nm very large granular vesicles; and (c) flattened or pleiomorphic agranular vesicles together with loo-nm granular vesicles. electron-dense Small (50 nm) vesicles containing material or terminals containing predominantly mitochondria were not encountered.
Glial Cells Enteric glial cells (Schwann’s cells] were numerous inside ganglia (Figure 5A) and accompanied secondary and tertiary elements with axons partly or completely invaginated in the cytoplasm (Figures 7A and 8). Glial cells had a conspicuous basal lamina on surfaces facing interstices. Intermediate filaments and microtubules were conspicuous cytoplasmic elements, and lipid droplets and secondary lysosomes
were sometimes encountered. Caveolae or dense bodies were never seen (Table 1). Fibroblastlike
Cells
These cells were less frequent than ICC in the intermuscular plane. They were identified in lowpower EM by their characteristically well-developed, often moderately dilated gER, which also extended into the processes. The long and slender shapes of FLC processes as seen commonly in sections can only be interpreted as profiles of sectioned broad and extremely flattened processes (Figures 3C and 6). Mitochondria were numerous, and the Golgi region was large. The nuclear chromatin was more condensed than in ICC or SMC (Figure 5A). The perinuclear cytoplasm was relatively scarce. No basal lamina, caveolae, or dense bodies were apparent. Intermediate filaments were visible, especially in the
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Pericytes (Figure 5C) were identified by their characteristic periendothelial location and by cytoplasmic (abundant caveolae) and nuclear (abundant heterochromatin) features. All structures between the main muscle layers were embedded in a “mat” of collagen fibers forming large loops and bundles, extending as smaller bundles inbetween all SMC of both layers (Figure 5A).
Discussion
Figure 11. Interstitial cells of Cajal processes (*) and SMC (M); ICC cytoplasm is packed with intermediate filaments. A basal lamina is present adjacent to dense bands (arrowbeads). Caveolae and cytoplasmic dense bodies are indicated (broad and thin arrows, respectively). E, elastin; C, collagen (original magnification x 15,000; bar = 1 pm).
processes. Coated vesicles and primary lysosomes were frequent. Lipid droplets were encountered, and a single cilium was occasionally present. Fibroblastlike cells established close contacts, some of which were of the intermediate type, with ICC. Gap junctions were not seen.
Other Cell Types Macrophagelike cells were identified by the irregular cytoplasmic processes, the abundance of primary and secondary lysosomes, and coated vesicles and coated pits (12). They were uncommon compared with ICC or FLC. Mast cells, eosinophils, and, occasionally, lymphocytes and plasma cells were also encountered.
Extracellular Space and Vessels Capillaries of the nonfenestrated type only, venules, and lymphatics were frequent between the muscle layers. Endothelial cells were distinguished from ICC by the abundance of caveolae, the welldeveloped basal lamina (Figure 5C) and the more slender processes, devoid of dense bodies and with less sER and fewer intermediate filaments (Table 1).
Our study provides evidence necessary for the interpretation of morphological or physiological studies on human small intestinal musculature. Interstitial cells of Cajal constitute an ultrastructurally distinct cell type, which on more cursory inspection may easily be confused especially with SMC. The distinction between ICC and SMC is very important. In a previous study (9), ICC associated with Auerbach’s plexus of human small intestine were claimed not to possess dense bodies, dense bands, or a basal lamina and to have only rare caveolae, whereas SMC in the same area were claimed to possess large cisternae of sER at the periphery. We have not been able to confirm this description. In the present study, ICC between the main muscle layers were clearly distinguishable from SMC of the same location but had several myoid features, i.e., they resembled modified smooth muscle cells as dense bodies, basal lamina (in certain places), caveolae; also, a particularly welldeveloped system of sER were typical ICC features. In contrast to SMC, thick (15 nm, myosin) filaments were never seen in ICC with the methods used; however, special techniques are necessary to document their absence. It is also important to distinguish between ICC and FLC. They are intimately associated with ICC and seem to occur as an integral part of ICC bundles. Fibroblastlike cells have been suggested to be involved in the coupling of longitudinal and circular muscle layers in the cat small intestine (13). We cannot exclude that human FLC constitute a part of an intestinal conduction system as suggested by the organization of the tissue. Only a limited number of EM studies have been performed on the human intestinal muscle coat (9,14-23). To date, ICC have not been included in ultrastructural studies on the pathology of the small intestine. The ultrastructural similarities between ICC and SMC suggest that ICC may also be excitable; further, ICC are seemingly intercalated between varicosities of nerve elements of Auerbach’s plexus and SMC or FLC of both main layers into which they extend. The organization of human small intestinal ICC in thick bundles provides further morphological evidence for the hypothesis that ICC may generate and/or propa-
GASTROENTEROLOGY
Figure 12. A. Interstitial cells of Cajal process surrounded by elastin material (E). The cytoplasm contains intermediate bodies (rig&frame, shown in B), and dense bands (lefrframe, shown in C) (original magnification x 10,000; bar = 1 km). B. Detail of cytoplasmic
dense bodies (DB) and intermediate
C. Detail of dense bands and basal lamina (arrowheads).
filaments (arrows)
(original magnification
x 73,000;
Arrow, coated vesicle; E, elastin (original magnification
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filaments,
dense
bar = 0.2 km). ~69,000;
bar = 0.2 km).
Figure 13. Bundle of ICC processes (*) split up by tertiary nerve elements containing naked axon terminals (A] with presynaptic specializations (arrowheads) (original magnification x 18,000; bar = 1 pm).
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Figure 14. Elastin fibers (E) surrounding ICC (*) and intervening between ICC and SMC (M) (original magnification ~26,000; bar = 0.5 km).
gate electric current or detect and modulate intestinal tone (l-6).The specific organization of ICC is equally compatible with a role in synchronization of electrical activity between the main muscle layers. There are many similarities between the structure and organization of the nodal cells in the sinus node of the heart (24) and human intestinal ICC, as shown by the present study. Nodal cells are also characterized by less organized filaments and a paucity of specialized intercellular junctions. Like ICC, nodal cells appose each other over large surface areas, separated by a rather constant gap of about 20 nm; gap junctions are not found along these clefts (24).Gap junctions were absent or inconspicuous also between ICC in the present study, as between SMC in the longitudinal muscle layer, but gap junctions may not be necessary for efficient electrical cell to cell coupling (25). Electrophysiological evidence from the small intestine of several species including humans (76) seems to favor two specific locations of pacemaker cells: the region between the main muscle layers and the region between the inner and outer subdivisions of the circular muscle layer. In humans, slow waves have been recorded from the longitudinal layer, only when the outer circular muscle is left attached (8), and from the outer part of the circular muscle in intact preparations only (8).These studies are compatible with our
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findings of a system of ICC extending from the outer parts of the circular layer to the inner parts of the longitudinal layer. However, microelectrode studies on human tissue are still preliminary (8). The size and cellular density of ICC in certain regions should make impalement with microelectrodes possible in intact preparations. The ultrastructure of ICC associated with Auerbath’s plexus may vary according to species; however, information is incomplete (Table 2). The myoid features of ICC are clearly most prominent in humans. Furthermore, in some species it seems to be a problem to distinguish the ultrastructure of FLC from that of ICC (13,26,27). AlthoughEM studies of sections using standard techniques cannot lead to identification or classification of all profiles encountered, it is evident that FLC and ICC are generally easily distinguishable as separate cell types. A more precise classification awaits the identification of specific immunologic markers. It is generally not possible to determine the transmitter contents of nerve terminals in routine EM preparations, but those with flattened vesicles seem to be noradrenergic terminals of the human small intestine (22). These terminals were not seen in close association with ICC, which were most often contacted by terminals with small, round agranular vesicles. A subpopulation of these may contain substance P (2 1).
Figure 15. Elastin fibers (E) and filaments (F) are located in grooves of ICC cytoplasm (*). Elastin filaments shows a distinct periodicity. Arrowhead, dense band with basal lamina material (original magnification X44,000; bar = 0.5 pm).
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Table 2. Comparative Ultrastructural Cytology of Interstitial Cells of Cajal Associated With Auerbach’s Plexus in the Small Intestine. Species Human Cat (13) Guinea pig (26, 28) Rat (28) Mouse (1,2,29)
Ca
Bm
+ + + + ++
sER
gER
Mi
DB
M
+ -
+++ -
(Z)
++ -
++ -
"
0 0 0
++ + +
++ ++ +++
0
+ (1)
NOTE. An approximation of relative frequency is indicated by pluses references are given in parentheses. -, Information not available; for abbreviations, see legend for Table 1.
The characteristic embedding of myenteric ganglia and primary fascicles in the longitudinal layer has not to our knowledge been described before in the small intestine. The smooth muscle cells encircling the ganglia might confer a key function in transmission, perhaps in synchronization of the muscle layers. There is a striking relationship between ICC and elastin fibers. The capacity of SMC (e.g., aortic) to produce elastin is well known, and it is possible that also ICC may produce elastin. The membrane properties of ICC might be affected by distention and stretch, and the elastin of the bowel wall could be relevant in this respect. The organization of elastin in the human intestine needs further study; elastin components could be important in various disease states. In conclusion, the present study of ICC associated with Auerbach’s plexus in the human small intestine has revealed a distinctive cell type conceived as a modified SMC with close relations to nerves, FLC, and SMC of both main layers. The cells are organized in bundles, their fine structure is compatible with the cells being excitable, and an important regulatory role of the cells is supported. The morphology of ICC as shown here provides a reference for future physiological and pathological studies of human small intestine. References 1. Thuneberg L. Interstitial cells of Cajal: intestinal pacemaker cells? Adv Anat Embryo1 Cell Biol1982;71:1-130. 2. Thuneberg L, Rumessen JJ, Mikkelsen HB. The interstitial cells of Cajal: intestinal pacemaker cells? In: Wienbeck M, ed. Motility of the digestive tract. New York: Raven, 1982:115122. 3. Rumessen JJ, Thuneberg L. Plexus muscularis profundus and associated interstitial cells. I. Light microscopical studies of mouse small intestine. Anat Ret 1982;203:115-127. 4. Rumessen JJ, Thuneberg L, Mikkelsen HB. Plexus muscularis profundus and associated interstitial cells. II. Ultrastructural studies of mouse small intestine. Anat Ret 1982;203:129-146. L. Interstitial cells of Cajal. In: Wood JD, ed. 5. Thuneberg Handbook of physiology. The gastrointestinal system. Volume 1. Bethesda, MD: American Physiological Society, 1989:349386. 6. Thuneberg L, Johansen V, Rumessen JJ, Andersen BG. Interstitial cells of Cajal: selective uptake of methylene blue inhibits
(+-+++).
7.
8.
9.
10. 11.
12.
13.
14. 15.
16.
17.
18.
19.
20.
21.
22.
0 or (+) means absent
I
A (+)
0
+++ + ++
0
++
+
or inconspicuous.
+
Relevant
slow wave activity. In: Roman C, ed. Gastrointestinal motility. Lancaster, England: MTP, 1984:495-502. Suzuki N, Prosser CL, Dahms V. Boundary cells between longitudinal and circular layers: essential for electrical slow waves in cat intestine. Am J Physiol1986;250:G287-G294. Hara Y, Kubota M, Szurszewski JH. Electrophysiology of smooth muscle of the small intestine of some mammals. J Physiol 1986;372:501-520. Faussone-Pellegrini MS, Cortesini C. Some ultrastructural features of the muscular coat of the human small intestine. Acta Anat 1983;115:47-68. Camilleri M, Phillips SF. Disorders of small intestinal motility. Gastroenterol Clin North Am 1989;18:405-424. Rgmert P, Matthiessen ME. Improved utilization of tissue blocks for electron microscopy. Effect of various concentrations of formaldehyde and glutaraldehyde. Acta Anat 1979;104: 388-393. Mikkelsen HB, Thuneberg L, Rumessen JJ, Thorball N. Macrophage-like cells in the muscularis externa of small intestine. Anat Ret 1985;213:77-86. Taylor AB, Kreulen D, Prosser CL. Electron microscopy of the connective tissues between longitudinal and circular muscle of small intestine of cat. Am JAnat 1977;150:427-442. Pick J, De Lemos C, Ciannella A. Fine structure of nerve terminals in the human gut. Anat Ret 1967;159:131-145. Greenberger NJ, Dobbins WO, Ruppert RD, Jesseph JE. Intestinal atony in progressive systemic sclerosis (scleroderma). Am J Med 1968;45:301-308. Dvorak AM, Osage JE, Monahan RA, Dickersin GR. Crohn’s disease: transmission electron microscopic studies. III. Target tissues. Proliferation of and injury to smooth muscle and the autonomic nervous system. Hum Path01 1980;11:620-634. Faussone-Pellegrini MS, Cortesini C. Ultrastructural peculiarities of the inner portion of the circular layer of colon. I. Research in the human. Acta Anat 1984;120:185-189. Shuffler MD, Lowe MC, Bill AH. Studies of idiopathic intestinal pseudoobstruction. I. Hereditary hollow visceral myopathy: clinical and pathological studies. Gastroenterology 1977; 73:327-338. Schuffler MD, Bird TD, Sumi SM, Cook A. A familiar neuronal disease presenting as intestinal pseudoobstruction. Gastroenterology 1978;75:889-898. Riemann JF, Schmidt H. Ultrastructural changes in the gut autonomic nervous system following laxative abuse and other conditions. Stand J Gastroenterol 1982;17(Suppl 71):111-124. Llwellyn-Smith IJ. Furness JB, Murphy R, O’Brien PE, Costa M. Substance P-containing nerves in the intestine. Distribution, ultrastructure and characterization of the immunoreactive peptide. Gastroenterology 1984;86:421-435. Llwellyn-Smith IJ, Furness JB, O’Brien PE, Costa M. Noradrenergic nerves in human small intestine. Distribution and ultrastructure. Gastroenterology 1984;87:513-529.
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23. Yoshida MM, Krishnamurthy S, Wattchow DA, Furness JB, Schuffler MD. Megacolon in myotonic dystrophy caused by a degenerative neuropathy of the myenteric plexus. Gastroenterology 1988;95:820-827. 24. Tranum-Jensen J. The fine structure of the sinus node: a survey. In: Bonk FIM, ed. The sinus node. Structure, function and clinical relevance. The Hague: Martinus Nijhoff, 1978:149165. 25. Daniel EE, Daniel VP, Duchon G, Garfield RE, Nichols M, Malhotra SK, Oki M. Is the nexus necessary for cell-to-cell coupling of smooth muscle? J Membrane Biol 1976;28:207239. 26. Cook RD, Burnstock G. The ultrastructure of Auerbach’s plexus in the guinea pig. II. Non-neuronal elements. J Neurocyto1 1976;5:195-206. 27. Komuro T. Three-dimensional observation of fibroblast-like cells associated with the rat myenteric plexus, with special reference to the interstitial cells of Cajal. Cell Tissue Res 1989;255:343-351.
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28. Gabella G. Innervation of the intestinal muscular coat. J Neurocytol 1972;1:341-362. 29. Yamamoto M. Electron microscopic studies on the innervation of the smooth muscle and the interstitial cell of Cajal in the small intestine of the mouse and bat. Arch Histol Jpn 1977;40: 171-201.
Received April 5, 1990. Accepted October 16, 1990. Address requests for reprints to: Jiiri Johannes Rumessen, M.D., Blegdamsvej 3, DK-2200 Copenhagen N, Denmark. This study was supported by grants from The Novo Foundation and Holger Hjortenbergs Fond. The authors are grateful to S. Peters, K. Stub-Christensen, and V. Heidemann for technical assistance and H. B. Mikkelsen for criticism of the manuscript. The helpful assistance of the staffs of the Department of Surgery D, Gentofte Hospital, and the Department of Surgery C, Rigshospitalet, is gratefully acknowledged.
1991;100:1417-1431
Interstitial Cells of Cajal in Human Small Intestine Ultrastructural Identification and Organization Between the Main Smooth Muscle Layers JCRI JOHANNES Anatomy Department
RUMESSEN C, University
and LARS THUNEBERG
of Copenhagen,
Previous morphological and electrophysiological studies have supported the hypothesis that interstitial cells of Cajal have important regulatory (pacemaker) functions in the gut. In the current study, interstitial cells of Cajal associated with Auerbach’s plexus in human small intestine were studied. Freshly resected intestine was examined by light and electron microscopy. The interstitial cells of Cajal resembled modified smooth muscle cells. They had caveolae and dense bodies, an incomplete basal lamina, a very well-developed smooth endoplasmic reticulum, and abundant intermediate (10nm) filaments. Myosin filaments were not seen. Fibroblastlike cells were distinguished by their lack of caveolae and dense bodies, the relative scarcity of smooth cisternae and intermediate filaments, and the abundant granular endoplasmic reticulum. Interstitial cells of Cajal were arranged in networks of bundles containing processes of two to seven cells with fibroblastlike cells interspersed in the bundles. The bundles were innervated by nerve elements of Auerbath’s plexus and extended into both layers of smooth muscle, between muscle cells, and into septa. The bundles were closely associated with elastin fibers. The organization shown in this study strongly supports the concept of interstitial cells of Cajal as important regulatory cells also in the human small intestine. The characteristic cytology and organization of interstitial cells of Cajal may provide a basis for future morphological, electrophysiological, and pathological studies of these cells in human small intestine.
0
ur
previous studies of the structural organization of interstitial cells of Cajal (ICC) located in the small intestine of the mouse led to the proposal that these cells had important regulatory functions and that a subpopulation of ICC located between the main
Panum Instituttet,
Copenhagen,
Denmark
muscle layers were ideal candidates for small intestinal pacemaker cells (l-5). Morphological and electrophysiological studies of several species including humans have supported this hypothesis (6-8). However, evidence of ICC in the human small intestine is scanty (9). Increasing attention is being given to motility disorders of the small intestine (10). Detailed knowledge of the structural and physiological characteristics of regulatory and slow wave-generating cell populations is therefore of fundamental importance and may give new insights into the control of gastrointestinal (GI) motility and changes in disease. We have therefore studied the ultrastructure of ICC located between the main muscle layers of the human small intestine. Materials
and Methods
Freshly resected, uninvolved small intestine (two duodenum, two jejunum, and seven terminal ileum) was obtained from nine adult patients (three female and six male; age, 32-90 years; median, 64 years) undergoing surgery because of incarceration of inguinal hernia or because of gastric, pancreatic, or colonic carcinoma. The patients had no other GI disease. They were not taking any drugs known to affect the structure of the enteric nerves or musculature. Patients with peritoneal carcinosis were excluded. After resection, l-Z-cm pieces of small intestine were immediately cut and immersed in two types of fixatives: (a) modified Karnovsky’s fixative (11) containing either 2% + 2%, 8% + 8%, or 12% + 12% glutaraldehyde (Taab, Berkshire, United Kingdom) and paraformaldehyde (Merck,
EM, electron microscopy; cells; gER, granular endoplasmic reticulum; ICC, interstitial cells of Cajal; sER, smooth endoplasmic reticulum; SMC, smooth muscle cells. o 1991 by the American Gastroenterological Association 0016-5085/91/$3.00 Abbreviations
FLC, fibroblastlike
used in this paper:
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sections were cut, mounted on copper grids, and contrasted with uranyl acetate and lead citrate. The grids were examined in Philips EM 300 and 400 electron microscopes (Eindhoven, The Netherlands). The study was performed in accordance with the Helsinki Declaration II and approved by the ethical committees of Copenhagen and Copenhagen County.
Results The muscle layers were thicker in the proximal small intestine, but the overall organization was similar in different regions (duodenum, jejunum, and ileum). Illustrations are mainly from the ileum, which was studied most extensively. Light Microscopy
Figure 1. Longitudinal section of the musculature in ileum. The circular layer (CM) consists of parallel laminae separated by connective tissue septa in continuity with the submucosa (SU). SE, serosa; LM, longitudinal muscle: long orrow, ganglion of Auerbach’s plexus: short arrow, subserous vessel with erythrocytes; arrowheads, innermost circular layer (toluidine blue; original magnification X 100; bar = 100 pm).
Darmstadt, Germany) in 0.1 mol/L phosphate buffer (Merck), pH 7.3, at 20°C for at least 2 hours; and (b) modified Bouin’s fixative containing 2% + 2% or 4% + 4% glutaraldehyde and paraformaldehyde in 0.1 mol/L phosphate buffer with picric acid (Merck) added from a saturated solution to give a final concentration of 0.1896, pH 7.3, at 20°C for at least 2 hours. After immersion, 1 x 2-mm pieces were cut and left in fixative overnight, rinsed for 60 minutes in 0.1 mol/L phosphate buffer, pH 7.3, and postfixed in 2% 0~0, in 0.1 mol/L phosphate buffer for 2 hours. The pieces were dehydrated in alcohol, block stained with alcoholic uranyl acetate, and embedded in Epon 812 R (Merck). Thin sections were stained with toluidine blue for light microscopical investigation, Suitable areas were selected for transmission electron microscopy [EM), and ultrathin (70 nm)
The general plan of the small intestinal muscularis externa is shown in Figure 1. The longitudinal muscle layer had a total thickness averaging 50-100 cells. The layer was subdivided by irregular connective tissue septa containing the larger nerves and vessels (Figure 1). Muscle cells with a similar orientation often surrounded ganglia and primary fascicles of the myenteric plexus, which were therefore partly or totally embedded in the longitudinal muscle layer (Figure 2) but never in the circular layer. In ganglionated areas, a large connective tissue space divided the main muscle layers, whereas in other areas the muscle layers were in close contact (Figure 2). The space contained smooth muscle cells (SMC), interstitial cells, vessels, and nerve elements of Auerbach’s plexus. Interstitial cell types of slightly different staining intensity were seen between the muscle layers, but specific cell types could not be identified with certainty (Figure 3A).
Figure 2. Ganglion (G) in Auerbach’s plexus surrounded by longitudinal muscle (Lhf), merging (arrows) with the circular layer (CM) (toluidine blue; original magnification x 240; bar = 50 nm).
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Figure 3. A. Septum (S) in longitudinal muscle layer (LM).Arrows, secondary nerve strands of Auerbach’s plexus; CM, circular muscle; upper frame, Figure 3C; lowerframe, Figure 3B; arrowheads, ICC processes (toluidine blue, original magnification x 800; bar = 10 pm). B. Lower frame in Figure 3A. Nucleus (N) and processes (*) of ICC located between the main muscle layers at the level of Auerbach’s plexus. Smooth cisternae are prominent in the processes (arrowheads). NF, tertiary nerve fascicles of Auerbach’s plexus (original magnification x 7500; bar = 2 km). C. Upper frame in Figure 3A. Processes (*) of ICC extending into septum between longitudinal muscle cells (LM). Processes of ICC are in intimate contact with two FLC (nucleus of the lower is indicated by N). M, SMC; NF, nerve fascicle (original magnification x 7500; bar = I km).
The circular muscle layer was divided by connective tissue septa in circularly oriented (ring-shaped) lamellae with a width of 50-150 pm running along the whole circumference (Figure 1). The septa were in continuity with the submucous layer and extended through the full thickness of the circular layer. A subdivision of the circular lamellae consisting of the innermost one to five cells was distinguishable in
most well-oriented sections, separated from the outer layer by a connective tissue space containing nerve elements of the deep muscular plexus, interstitial cells, and vessels. The innermost subdivision of the circular layer could be distinguished as covering the inner one fourth to one third of the muscular lamellae, after which it seemed to merge with the bulk of the circular lamellae.
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Smooth Muscle Cells
Figure 4. Longitudinally sectioned SMC (M) and ICC process (*). Interstitial Cells of Cajal cytoplasm contains irregularly arranged intermediate filaments (IF), and a few dense bodies are seen (arrowheads). Myosin filaments and basal lamina are absent in contrast to SMC, in which myosin is depicted by arrowheads and dense bodies by LUTOWS(original magnification ~15,000; bar =
In low-power EM, SMC were often impossible to distinguish from ICC (see later). At higher magnification, SMC of both layers were easily distinguished by their size, arrangement, and in particular their characteristic arrangement of dense bodies and conspicuous myosin filaments (Figure 4). Caveolae were frequent, arranged in longitudinal bands alternating with dense bands (Figures 5B and 6). Cytoplasmic dense bodies were very conspicuous and regularly distributed (Figure 4). Submembraneous smooth endoplasmic reticulum (sER) and rows of mitochondria were characteristically located close to the caveolated membrane areas. Other organelles were concentrated in the larger perinuclear regions. The nucleus had sparse, peripherally distributed het,erochromatin (Figure 5A). The SMC surrounding the ganglia and larger fascicles of Auerbach’s plexus had the same ultrastructural features. Muscle cells in the circular layer were interconnected by adherens-type and gap junctions. The latter were not seen in the longitudinal layer. No mitoses were seen in SMC. Nerve terminals were only occasionally in close, synapselike contact with SMC of either layer (Figure 6). The characteristic features are summarized in Table 1. Interstitial Cells of Cajal
1 w).
Iden tifca tion The outer, main layer of circular muscle was about twice the thickness of the longitudinal muscle layer and generally comprised 150-200 cells in the ileum. Both the longitudinal and circular layers were pierced by smaller connective tissue septa containing capillaries and tertiary nerve elements accompanied by interstitial cell types. The larger connecting nerve strands and vessels ran in the septa between the circular muscle lamellae.
The problem of identifying ICC in sections is best illustrated by considering Figures 5A, 7A, and 8. In these low-power electron micrographs ICC may be difficult to distinguish from fibroblastlike cells (FLC) and especially from SMC. The distinctive cytological features are shown at higher magnifications (Figures 5B and C and 7B and C), in which the abundance of intermediate filaments and sER is most characteristic. Caveolae, dense bodies, and basal lamina are variably developed.
Electron Microscopy The ultrastructural preservation of the superficial parts of the musculature (from serosa to the outer parts of the circular muscle) was generally satisfactory and allowed detailed cytological studies with all fixatives used. Axonal varicosities and neuronal mitochondria were often suboptimally preserved, especially in the deeper layers of the circular muscle, whereas muscle and interstitial cells were well preserved at corresponding levels. The tissue in the deep layers was better preserved when the fixative with the highest aldehyde concentrations was used. Glycogen granules were most prominent in the modified Bouin’sfixed material.
Cytological Features Light microscopy. In toluidine blue-stained sections, cells with abundant, lightly stained cytoplasm and an ovoid nucleus with a finely granular chromatin were visible (Figure 3A). The nuclear features did not suffice to distinguish ICC from other interstitial elements, and their processes were very similar .to SMC processes (Figure 3A). Electron microscopy. Profiles of ICC processes were long with only little apparent branching (Figure 5A). Processes of ICC were narrower and of more heterogeneous size than those of SMC (Figures 4 and 5A). Organelles seemed heterogeneously distributed
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region in ileum. Interstitial cells of Cajal processes (*) partially Figm re 5. A. Survey of the intermuscular form bundles extending into the longitudinal muscle layer (LM). Frames b and c are magnified in Figure x 2800; bar = 10 km). (Figure continues on page 1422.) cell (original magnification
in ICC processes, which were dominated by a very well-developed sER in broad peripheral zones (Figures 3B and 5B) and a dense network of intermediate (10 nm) filaments (Figures 4 and 7B). Intermediate filaments were organized in bundles, more parallel in the smaller processes. Other (few) ICC processes were dominated by large mitochondria with flattened cristae (Figure 7C). Microtubules were often seen; granular endoplasmic reticulum (gER) was inconspicuous. Glycogen granules were most often evenly distributed within processes, and they had a density and size comparable to those in SMC. Some ICC processes were packed with clumps of glycogen (Figure 9). The perinuclear regions of ICC (Figures 8 and 10) had abundant cytoplasm with a crowding of gER and Golgi stacks but contained less sER than ICC pro-
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ensheath nerve elements (NF] I and 4B and C. F, FLC; End, endotb elial
cesses. Intermediate filaments were also abundant in perinuclear regions (Figure 10). The ICC nuclei (Figures 3B and 10) had a chromatin pattern similar to SMC nuclei, and the contours were often rather irregular. The ICC had a variably developed basal lamina, most prominent in regions of dense bands and in some ICC located inside the muscle layers (Figures 7C and 11). Caveolae were distinctive, of similar appearance as in SMC, but clearly fewer in number (Figures 10 and 11). Caveolae alternated only at times with dense bands (Figure 11); a clear distinctive feature, as opposed to SMC, was the relatively few dense bands (Figures 4 and 5B). The ICC cytoplasm contained conspicuous dense bodies, although smaller and more irregularly distributed than in SMC (Figures 11 and 12).
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Figure 5 (cont’d.). B. Bundle of 5 (l-5)ICCprocesses dominated by sER (arrowheads) and intermediate filaments. NF, tertiary nerve elements; M, SMC in longitudinal layer; End, endothelial cell (original magnification X 12,000; bar = 1 pm). C. Cross-sectioned ICC bundle (*) and a single SMC (M). At the bottom a pericyte (P) and an endothelial cell (End) are seen. NF, tertiary nerve fascicle; N, nucleus of ICC (original magnification X8500; bar = 2 km).
Thick (15 nm, myosin) filaments were not seen in ICC in sharp contrast to the abundance of myosin filaments in SMC (Figure 4). Thin filaments (5 nm, actin) were rare, mostly located in peripheral zones of ICC cytoplasm. Another characteristic (although not specific) feature of ICC was the occurrence of lipid droplets (Figure 10). These droplets were either homogeneous and moderately electron dense, or they were more heterogeneous. A unit membrane could not be dis-
cerned. Occasionally a single cilium was seen. Coated vesicles and free ribosomes were frequent [Figures 10 and 12C). The cytology of ICC compared with other cell types in the same area is summarized in Table 1. Organization Processes of ICC were very abundant in ganglionated areas, distributed all over the intermuscular
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plane within the dense collagen mat (Figure 5A). The ICC formed incomplete sheaths around ganglia and smaller fascicles and were mostly arranged in groups and bundles of three to seven cells with large areas of membrane closely apposed and separated by a rather constant cleft of about 20 nm (Figure 5B). The density and size of ICC bundles seemed greatest in the duodenum. Adherens-type junctions between ICC were seen, but gap junctions were not encountered. The ICC bundles extended from the intermuscular plane into the longitudinal layer (Figures 3C and 5A) or into the circular layer (Figure 7). The ICC penetrated both layers into the main septa (Figures 3 and 7) as well as in between SMC (Figure 5A). Gap junctions between ICC and other cells including SMC were not seen. The ICC bundles were always in intimate contact with nerve elements of Auerbach’s plexus, and synaptic specializations with clustering of vesicles and presynaptic densities were frequent (Figures 8 and 13). These varicosities contained either small 50-nm agranular vesicles together with 100-nm granular vesicles or 50-nm agranular vesicles together with large (150-ZOO-nm) dense core vesicles. The ICC were often in intimate contact with FLC, and adherenstype (intermediate) junctions were occasionally seen (Figures 3C and 6). Fibroblastlike cells were interspersed in the ICC bundles and seemed to represent an extension of some of these in the deeper parts of both muscle layers (Figure 6). Close relations to macrophagelike cells were also seen but infrequently. The extracellular elastin had a typical and rather consistent localization and relation to ICC bundles (Figures 14 and 15). Elastin material in different stages of elastogenesis (fibrils and small or larger homogeneous fibers) were nearly always scattered around individual ICC, often in close contact with the plasma membrane (Figure 15). Elastin was located between individual ICC and between ICC and FLC or SMC (Figure 14). This arrangement suggested a more specific relation to ICC bundles and processes. Elastic fiber microfibrils were 10-15 nm long and had a distinct periodicity (Figures 12C and 15).
Nerve Elements
Figure 6. Connections between ICC (*) and FLC (short arrows) as well as FLC-SMC (long arrows) in the longitudinal muscle layer (M). Arrowheads, axon terminal in synapselike contact with SMC; NF, nerve fascicle; E, elastin (original magnification X 10,000; bar = 2 pm).
Ganglia and primary fascicles of Auerbach’s plexus were closely apposed to or embedded in the longitudinal muscle layer (Figures 1 and 2). Secondary and tertiary elements ramified in both muscle layers as well as in the intermuscular plane and connective tissue septa. Tertiary elements (3-15 axons) followed processes of ICC closely. Three main types of nerve varicosities were distinguished based on their content of vesicles (Figures 8 and 13): (a) 50-nm small agranular vesicles together with 100-nm
Table 1. Distinctive Cytological Features of Different Cell Types Associated With Auerbach’s in the Human Small Intestine. Cell type ICC cell bodies ICC processes SMC FLC MLC Glial cell Endothelial cell
Ca
Bm
sER
gER
++ ++ +++ 0 0 0 +++
+ + +++ 0 0 +++ +++
++ +++ ++ + + + +
(1, + +++ + + +
DB
M
I
A
Li
+
+
0
++
0 0
++ +++
+ +
++ ++
+ ++ +++ + +
+++ 0 0 0 0
+++ 0 0 0 0
Mi
cv
LY
++
++
+ ++ ++ + + +
(1, + +++ 0 0
Plexus
++ + + ++ +
+++
+
(+I (+I 0 0
+ 0 + 0
NOTE. An approximation of relative frequency is indicated by pluses (+-+ + +); 0 or (+) means not encountered or inconspicuous. Ca, caveola; Bm, basal lamina; Mi, mitochondria; Cv, coated vesicles; Ly, lysosomes; DB, dense bands/bodies; M, thick filaments; intermediate filament: A, thin filaments: Li, lipid droplets.
I,
Figure 7. A. Interstitial cells of Cajal processes (*) running in a septum between circular muscle cells (CM]. Gc, glial cell with nerve profiles; frames, detailed in B and C; IM, intermuscular plane (original magnification x 6000; bar = z urn). B. Upper frame in A, neighboring section. The ICC cytoplasm is dominated by intermediate filaments (IF). Broad arrow, FLC process; arrowheatl, dense band (original magnification ~13,000; bar =
1 km). C. Lower frame in A, neighboring section. This ICC is dominated by numerous large mitochondria (Mi) and a continuous basal lamina is apparent (arrowheads). M, muscle cells (original magnification ~14,000; bar = 1 km).
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Figure 8. Organization of ICC processes (*) and smaller nerves (NF) between a ganglion (G) and the longitudinal muscle 1~ iyer (L14. 1nterstiti: 11cells of Cajal processes are in close contact with a tertiary nerve fascicle (NF) with a naked terminal containing lauge der we core vf !sic:les (long arrow). Short arrows, process of an FLC; M, SMC; E, elastin (original magnification x 12,500; bar = 1 pm).
Figure 9. Interstitial cells of Cajal processes (*) as they appear after fixation with a fixative containing picrate (modified Bo uin’ Glycog en granules are well preserved (arrows). The heterogeneous distribution within and in between different processes is aplparent. E, elastin fibIrils; * , ICC processes; C, collagen fibrils (original magnification x 22,500; bar = 1 urn).
a.
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Figure 10. P&nuclear region of ICC showing dense loops and bundles of intermediate filaments (IF) with regional clusteri ng of mitoch .ondria (Mi) and gER.Caveolae are indicated (arrowheads). M, SMC; N, ICC nucleus; arrow, lipid droplets with flocr :ulent al. *Process of another ICC (original magnification X 14,000; bar = 1 km).
large dense core vesicles; (b) 50-nm small agranular vesicles together with 150~200-nm very large granular vesicles; and (c) flattened or pleiomorphic agranular vesicles together with loo-nm granular vesicles. electron-dense Small (50 nm) vesicles containing material or terminals containing predominantly mitochondria were not encountered.
Glial Cells Enteric glial cells (Schwann’s cells] were numerous inside ganglia (Figure 5A) and accompanied secondary and tertiary elements with axons partly or completely invaginated in the cytoplasm (Figures 7A and 8). Glial cells had a conspicuous basal lamina on surfaces facing interstices. Intermediate filaments and microtubules were conspicuous cytoplasmic elements, and lipid droplets and secondary lysosomes
were sometimes encountered. Caveolae or dense bodies were never seen (Table 1). Fibroblastlike
Cells
These cells were less frequent than ICC in the intermuscular plane. They were identified in lowpower EM by their characteristically well-developed, often moderately dilated gER, which also extended into the processes. The long and slender shapes of FLC processes as seen commonly in sections can only be interpreted as profiles of sectioned broad and extremely flattened processes (Figures 3C and 6). Mitochondria were numerous, and the Golgi region was large. The nuclear chromatin was more condensed than in ICC or SMC (Figure 5A). The perinuclear cytoplasm was relatively scarce. No basal lamina, caveolae, or dense bodies were apparent. Intermediate filaments were visible, especially in the
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Pericytes (Figure 5C) were identified by their characteristic periendothelial location and by cytoplasmic (abundant caveolae) and nuclear (abundant heterochromatin) features. All structures between the main muscle layers were embedded in a “mat” of collagen fibers forming large loops and bundles, extending as smaller bundles inbetween all SMC of both layers (Figure 5A).
Discussion
Figure 11. Interstitial cells of Cajal processes (*) and SMC (M); ICC cytoplasm is packed with intermediate filaments. A basal lamina is present adjacent to dense bands (arrowbeads). Caveolae and cytoplasmic dense bodies are indicated (broad and thin arrows, respectively). E, elastin; C, collagen (original magnification x 15,000; bar = 1 pm).
processes. Coated vesicles and primary lysosomes were frequent. Lipid droplets were encountered, and a single cilium was occasionally present. Fibroblastlike cells established close contacts, some of which were of the intermediate type, with ICC. Gap junctions were not seen.
Other Cell Types Macrophagelike cells were identified by the irregular cytoplasmic processes, the abundance of primary and secondary lysosomes, and coated vesicles and coated pits (12). They were uncommon compared with ICC or FLC. Mast cells, eosinophils, and, occasionally, lymphocytes and plasma cells were also encountered.
Extracellular Space and Vessels Capillaries of the nonfenestrated type only, venules, and lymphatics were frequent between the muscle layers. Endothelial cells were distinguished from ICC by the abundance of caveolae, the welldeveloped basal lamina (Figure 5C) and the more slender processes, devoid of dense bodies and with less sER and fewer intermediate filaments (Table 1).
Our study provides evidence necessary for the interpretation of morphological or physiological studies on human small intestinal musculature. Interstitial cells of Cajal constitute an ultrastructurally distinct cell type, which on more cursory inspection may easily be confused especially with SMC. The distinction between ICC and SMC is very important. In a previous study (9), ICC associated with Auerbach’s plexus of human small intestine were claimed not to possess dense bodies, dense bands, or a basal lamina and to have only rare caveolae, whereas SMC in the same area were claimed to possess large cisternae of sER at the periphery. We have not been able to confirm this description. In the present study, ICC between the main muscle layers were clearly distinguishable from SMC of the same location but had several myoid features, i.e., they resembled modified smooth muscle cells as dense bodies, basal lamina (in certain places), caveolae; also, a particularly welldeveloped system of sER were typical ICC features. In contrast to SMC, thick (15 nm, myosin) filaments were never seen in ICC with the methods used; however, special techniques are necessary to document their absence. It is also important to distinguish between ICC and FLC. They are intimately associated with ICC and seem to occur as an integral part of ICC bundles. Fibroblastlike cells have been suggested to be involved in the coupling of longitudinal and circular muscle layers in the cat small intestine (13). We cannot exclude that human FLC constitute a part of an intestinal conduction system as suggested by the organization of the tissue. Only a limited number of EM studies have been performed on the human intestinal muscle coat (9,14-23). To date, ICC have not been included in ultrastructural studies on the pathology of the small intestine. The ultrastructural similarities between ICC and SMC suggest that ICC may also be excitable; further, ICC are seemingly intercalated between varicosities of nerve elements of Auerbach’s plexus and SMC or FLC of both main layers into which they extend. The organization of human small intestinal ICC in thick bundles provides further morphological evidence for the hypothesis that ICC may generate and/or propa-
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Figure 12. A. Interstitial cells of Cajal process surrounded by elastin material (E). The cytoplasm contains intermediate bodies (rig&frame, shown in B), and dense bands (lefrframe, shown in C) (original magnification x 10,000; bar = 1 km). B. Detail of cytoplasmic
dense bodies (DB) and intermediate
C. Detail of dense bands and basal lamina (arrowheads).
filaments (arrows)
(original magnification
x 73,000;
Arrow, coated vesicle; E, elastin (original magnification
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filaments,
dense
bar = 0.2 km). ~69,000;
bar = 0.2 km).
Figure 13. Bundle of ICC processes (*) split up by tertiary nerve elements containing naked axon terminals (A] with presynaptic specializations (arrowheads) (original magnification x 18,000; bar = 1 pm).
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Figure 14. Elastin fibers (E) surrounding ICC (*) and intervening between ICC and SMC (M) (original magnification ~26,000; bar = 0.5 km).
gate electric current or detect and modulate intestinal tone (l-6).The specific organization of ICC is equally compatible with a role in synchronization of electrical activity between the main muscle layers. There are many similarities between the structure and organization of the nodal cells in the sinus node of the heart (24) and human intestinal ICC, as shown by the present study. Nodal cells are also characterized by less organized filaments and a paucity of specialized intercellular junctions. Like ICC, nodal cells appose each other over large surface areas, separated by a rather constant gap of about 20 nm; gap junctions are not found along these clefts (24).Gap junctions were absent or inconspicuous also between ICC in the present study, as between SMC in the longitudinal muscle layer, but gap junctions may not be necessary for efficient electrical cell to cell coupling (25). Electrophysiological evidence from the small intestine of several species including humans (76) seems to favor two specific locations of pacemaker cells: the region between the main muscle layers and the region between the inner and outer subdivisions of the circular muscle layer. In humans, slow waves have been recorded from the longitudinal layer, only when the outer circular muscle is left attached (8), and from the outer part of the circular muscle in intact preparations only (8).These studies are compatible with our
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findings of a system of ICC extending from the outer parts of the circular layer to the inner parts of the longitudinal layer. However, microelectrode studies on human tissue are still preliminary (8). The size and cellular density of ICC in certain regions should make impalement with microelectrodes possible in intact preparations. The ultrastructure of ICC associated with Auerbath’s plexus may vary according to species; however, information is incomplete (Table 2). The myoid features of ICC are clearly most prominent in humans. Furthermore, in some species it seems to be a problem to distinguish the ultrastructure of FLC from that of ICC (13,26,27). AlthoughEM studies of sections using standard techniques cannot lead to identification or classification of all profiles encountered, it is evident that FLC and ICC are generally easily distinguishable as separate cell types. A more precise classification awaits the identification of specific immunologic markers. It is generally not possible to determine the transmitter contents of nerve terminals in routine EM preparations, but those with flattened vesicles seem to be noradrenergic terminals of the human small intestine (22). These terminals were not seen in close association with ICC, which were most often contacted by terminals with small, round agranular vesicles. A subpopulation of these may contain substance P (2 1).
Figure 15. Elastin fibers (E) and filaments (F) are located in grooves of ICC cytoplasm (*). Elastin filaments shows a distinct periodicity. Arrowhead, dense band with basal lamina material (original magnification X44,000; bar = 0.5 pm).
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Table 2. Comparative Ultrastructural Cytology of Interstitial Cells of Cajal Associated With Auerbach’s Plexus in the Small Intestine. Species Human Cat (13) Guinea pig (26, 28) Rat (28) Mouse (1,2,29)
Ca
Bm
+ + + + ++
sER
gER
Mi
DB
M
+ -
+++ -
(Z)
++ -
++ -
"
0 0 0
++ + +
++ ++ +++
0
+ (1)
NOTE. An approximation of relative frequency is indicated by pluses references are given in parentheses. -, Information not available; for abbreviations, see legend for Table 1.
The characteristic embedding of myenteric ganglia and primary fascicles in the longitudinal layer has not to our knowledge been described before in the small intestine. The smooth muscle cells encircling the ganglia might confer a key function in transmission, perhaps in synchronization of the muscle layers. There is a striking relationship between ICC and elastin fibers. The capacity of SMC (e.g., aortic) to produce elastin is well known, and it is possible that also ICC may produce elastin. The membrane properties of ICC might be affected by distention and stretch, and the elastin of the bowel wall could be relevant in this respect. The organization of elastin in the human intestine needs further study; elastin components could be important in various disease states. In conclusion, the present study of ICC associated with Auerbach’s plexus in the human small intestine has revealed a distinctive cell type conceived as a modified SMC with close relations to nerves, FLC, and SMC of both main layers. The cells are organized in bundles, their fine structure is compatible with the cells being excitable, and an important regulatory role of the cells is supported. The morphology of ICC as shown here provides a reference for future physiological and pathological studies of human small intestine. References 1. Thuneberg L. Interstitial cells of Cajal: intestinal pacemaker cells? Adv Anat Embryo1 Cell Biol1982;71:1-130. 2. Thuneberg L, Rumessen JJ, Mikkelsen HB. The interstitial cells of Cajal: intestinal pacemaker cells? In: Wienbeck M, ed. Motility of the digestive tract. New York: Raven, 1982:115122. 3. Rumessen JJ, Thuneberg L. Plexus muscularis profundus and associated interstitial cells. I. Light microscopical studies of mouse small intestine. Anat Ret 1982;203:115-127. 4. Rumessen JJ, Thuneberg L, Mikkelsen HB. Plexus muscularis profundus and associated interstitial cells. II. Ultrastructural studies of mouse small intestine. Anat Ret 1982;203:129-146. L. Interstitial cells of Cajal. In: Wood JD, ed. 5. Thuneberg Handbook of physiology. The gastrointestinal system. Volume 1. Bethesda, MD: American Physiological Society, 1989:349386. 6. Thuneberg L, Johansen V, Rumessen JJ, Andersen BG. Interstitial cells of Cajal: selective uptake of methylene blue inhibits
(+-+++).
7.
8.
9.
10. 11.
12.
13.
14. 15.
16.
17.
18.
19.
20.
21.
22.
0 or (+) means absent
I
A (+)
0
+++ + ++
0
++
+
or inconspicuous.
+
Relevant
slow wave activity. In: Roman C, ed. Gastrointestinal motility. Lancaster, England: MTP, 1984:495-502. Suzuki N, Prosser CL, Dahms V. Boundary cells between longitudinal and circular layers: essential for electrical slow waves in cat intestine. Am J Physiol1986;250:G287-G294. Hara Y, Kubota M, Szurszewski JH. Electrophysiology of smooth muscle of the small intestine of some mammals. J Physiol 1986;372:501-520. Faussone-Pellegrini MS, Cortesini C. Some ultrastructural features of the muscular coat of the human small intestine. Acta Anat 1983;115:47-68. Camilleri M, Phillips SF. Disorders of small intestinal motility. Gastroenterol Clin North Am 1989;18:405-424. Rgmert P, Matthiessen ME. Improved utilization of tissue blocks for electron microscopy. Effect of various concentrations of formaldehyde and glutaraldehyde. Acta Anat 1979;104: 388-393. Mikkelsen HB, Thuneberg L, Rumessen JJ, Thorball N. Macrophage-like cells in the muscularis externa of small intestine. Anat Ret 1985;213:77-86. Taylor AB, Kreulen D, Prosser CL. Electron microscopy of the connective tissues between longitudinal and circular muscle of small intestine of cat. Am JAnat 1977;150:427-442. Pick J, De Lemos C, Ciannella A. Fine structure of nerve terminals in the human gut. Anat Ret 1967;159:131-145. Greenberger NJ, Dobbins WO, Ruppert RD, Jesseph JE. Intestinal atony in progressive systemic sclerosis (scleroderma). Am J Med 1968;45:301-308. Dvorak AM, Osage JE, Monahan RA, Dickersin GR. Crohn’s disease: transmission electron microscopic studies. III. Target tissues. Proliferation of and injury to smooth muscle and the autonomic nervous system. Hum Path01 1980;11:620-634. Faussone-Pellegrini MS, Cortesini C. Ultrastructural peculiarities of the inner portion of the circular layer of colon. I. Research in the human. Acta Anat 1984;120:185-189. Shuffler MD, Lowe MC, Bill AH. Studies of idiopathic intestinal pseudoobstruction. I. Hereditary hollow visceral myopathy: clinical and pathological studies. Gastroenterology 1977; 73:327-338. Schuffler MD, Bird TD, Sumi SM, Cook A. A familiar neuronal disease presenting as intestinal pseudoobstruction. Gastroenterology 1978;75:889-898. Riemann JF, Schmidt H. Ultrastructural changes in the gut autonomic nervous system following laxative abuse and other conditions. Stand J Gastroenterol 1982;17(Suppl 71):111-124. Llwellyn-Smith IJ. Furness JB, Murphy R, O’Brien PE, Costa M. Substance P-containing nerves in the intestine. Distribution, ultrastructure and characterization of the immunoreactive peptide. Gastroenterology 1984;86:421-435. Llwellyn-Smith IJ, Furness JB, O’Brien PE, Costa M. Noradrenergic nerves in human small intestine. Distribution and ultrastructure. Gastroenterology 1984;87:513-529.
May 1991
23. Yoshida MM, Krishnamurthy S, Wattchow DA, Furness JB, Schuffler MD. Megacolon in myotonic dystrophy caused by a degenerative neuropathy of the myenteric plexus. Gastroenterology 1988;95:820-827. 24. Tranum-Jensen J. The fine structure of the sinus node: a survey. In: Bonk FIM, ed. The sinus node. Structure, function and clinical relevance. The Hague: Martinus Nijhoff, 1978:149165. 25. Daniel EE, Daniel VP, Duchon G, Garfield RE, Nichols M, Malhotra SK, Oki M. Is the nexus necessary for cell-to-cell coupling of smooth muscle? J Membrane Biol 1976;28:207239. 26. Cook RD, Burnstock G. The ultrastructure of Auerbach’s plexus in the guinea pig. II. Non-neuronal elements. J Neurocyto1 1976;5:195-206. 27. Komuro T. Three-dimensional observation of fibroblast-like cells associated with the rat myenteric plexus, with special reference to the interstitial cells of Cajal. Cell Tissue Res 1989;255:343-351.
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28. Gabella G. Innervation of the intestinal muscular coat. J Neurocytol 1972;1:341-362. 29. Yamamoto M. Electron microscopic studies on the innervation of the smooth muscle and the interstitial cell of Cajal in the small intestine of the mouse and bat. Arch Histol Jpn 1977;40: 171-201.
Received April 5, 1990. Accepted October 16, 1990. Address requests for reprints to: Jiiri Johannes Rumessen, M.D., Blegdamsvej 3, DK-2200 Copenhagen N, Denmark. This study was supported by grants from The Novo Foundation and Holger Hjortenbergs Fond. The authors are grateful to S. Peters, K. Stub-Christensen, and V. Heidemann for technical assistance and H. B. Mikkelsen for criticism of the manuscript. The helpful assistance of the staffs of the Department of Surgery D, Gentofte Hospital, and the Department of Surgery C, Rigshospitalet, is gratefully acknowledged.