GASTROENTEROLOGY
1992:102:56-68
Ultrastructure of Interstitial Cells of Cajal Associated With Deep Muscular Plexus of Human Smal1 Intestine JÜRI JOHANNES RUMESSEN, and LARS THUNEBERG
HANNE
BIRTE
MIKKELSEN,
Department of Anatomy C, University of Copenhagen, Panum Instituttet, Copenhagen, Denmark
Evidente showing that interstitial cells of Cajal have important regulatory functions in the gut musculature is accumulating. In the current study, the ultrastructure of the deep muscular plexus and associated interstial cells of Cajal in human smal1 intestine were studied to provide a reference for identification and further physiological or pathological studies. The deep muscular plexus was sandwiched between a thin inner layer of smooth muscle (one to five cells thick) and the bulk of the circular muscle. Interstitial cells of Cajal in this region very much resembled smooth muscle cells (with a continuous basal lamina, caveolae, intermediate filaments, dense bodies, dense bands, and a well-developed subsurface smooth endoplasmic reticulum), but the arrangement of organelles was clearly different, and cisternae of granular endoplasmic reticulum were abundant. Interstitial cells of Cajal were distinguished from fibroblasts or macrophages in the region. They ramified in the inner zone of the outer division of circular muscle, penetrated the innermost circular layer, and were also found at the submucosal border. They were in close, synapselike contact with nerve terminals of the deep muscular plexus, and only few gap junctions with other interstitial cells of Cajal or with the musculature were observed. Compared with interstitial cells of Cajal from other mammals, those associated with the deep muscular plexus in the human smal1 intestine more closely resemble smooth muscle cells, and their organization appears more diffuse; however, the ultrastructure and organization of interstitial cells of Cajal is compatible with modulatory actions on the circular muscle also in humans.
1
n the smal1 intestinal muscularis externa, the deep muscular plexus (DMP or plexus muscularis profundus) occupies a narrow space between the thick outer and thin inner layer of circular muscle cells.l-‘o The potential importante of DMP as an intrinsic neural regulator of circular muscle function is becoming increasingly clear.“-‘3 Slow waves of the
smal1 intestine may, in part, originate in nonneural cells located between the two subdivisions of the circular muscle layer.14 The exact cellular origin has not been established. Ultrastructural studies of this region in different animal species have focused on the importante of interstitial cells of Cajal (ICC) as myoid cells intercalated between the DMP and the circular musc1e.6-g,‘5*‘6Based on this, a key role of ICC-DMP as regulators of circular muscle function has been strongly suggested. The ultrastructure of DMP and associated ICC in human smal1 intestine has been studied very little.17 In the present study we extend our previous observations of ICC in mouse DMP7-’ to human smal1 intestine, with emphasis on a clear identification of ICC and their detailed ultrastructural organization. In conjunction with our recent study of ICC in the region between the main muscle layers,18 the present results may provide a framework for future electrophysiological as wel1 as pathologica1 studies of human smal1 intestine. Materials and Methods Samples of freshly resected, uninvolved smal1 intestine (2 from the duodenum, 2 from the jejunum, and 10 from the ileum) were obtained from 12 adult patients (3 women and 9 men, aged 27-90 years; median, 60 years) undergoing surgery because of incarceration of inguinal hernia or gastric, pancreatic, or colonic malignancies. The patients had no other gastrointestinal disease, and patients with peritoneal carcinosis were excluded. The tissue was handled and processed for transmission electronmicroscopy (Philips EM 300 and 400; Eindhoven, The Netherlands) as previously described.” After resection, l-2-cm pieces of smal1 intestine were immediately cut and immersed in the following two fixatives: (a) a modified Karnovsky fixative” containing 2% + 2%, 8% + 8%, or 12% + 12% glutaraldehyde (Taab, Berkshire, England) and paraformaldehyde (Merck, Darmstadt, Germany) in 0.1 mol/L phosphate buffer (Merck), pH 7.3, at 20°C for at 0 1992 by the American Gastroenterological 0016-5085/92/$3.00
Association
January 1992
INTERSTITIAL CELLS OF CAJAL IN THE DEEP MUSCULAR
PLEXUS
57
Figure 1. Circular muscle lamina in cross-section. Deep muscular plexus is distinctly located between tbe thin innermost layer of circular muscle cells (arrows) and the outer bulk of circular muscle (OCM) but fades out towards Auerbach’s plexus (arrowheads). S, septa between circular muscle laminae; SU, submucosa. (Toluidine blue; original magnification X260; bar = 50 pm.) Figure 2. Innermost zone of the circular muscle. The innermost layer (arrows) deviates (OCM) (toluidine blue, original magnification X400; bar = 50 pm).
from the circular
direction
of the outer layer
Figures 3 and 4. Cross-section (Figure 3) and tangential (Figure 4) section of DMP and associated interstitial elements as they appear in routine light microscopical sections stained with toluidine blue. Identification of different cel1 types labeled here requires electron microscopy at higher magnifications (sec later figures). Arrow, ICC; F, FLC; M, MLC; S, glial cell; MC, mast cell; N, nerve fascicle; C, capillary; Su, submucosa; OCM, outer circular layer; KM, inner circular layer. [Original magnifications: X960, bar = 20 pm (Figure 3); X1050, bar = 20 pm (Figure 4).]
58
RUMESSEN ET AL.
GASTROENTEROLOGY Vol. 102, No. 1
Figure 5. Interstitial cells of Cajal (*) ramifying in the inner zone of the outer circular layer (M). ICM, inner circular layer. (Original magnification X9999; bar = 2 pm.) Figure 6. Interstitial cells of Cajal (*) contacting two muscle cells and bridging the two circular layers. OCM, outer circular layer; ICM, inner circular layer. (Original magnification X17,800; bar = 1 Pm.1
least z hours; and (b) a modified Bouin 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 0.18%, 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% OsO, in 0.1 mol/ L phosphate buffer for 2 hours. The pieces were dehydrated in alcohol, black-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, and ultrathin (70 nm) sections were cut, mounted on copper grids, and contrasted with uranyl acetate and lead citrate. The study was performed in accordance with the Helsinki Declaration 11 and approved by the ethica1 committees of Copenhagen and Copenhagen County.
Results We found no differences in the principal arrangement of ICC and DMP in the different parts of
the smal1 intestine. Light Microscopy
of Deep Muscular Plexus
The deep muscular plexus was sandwiched between a thin (one to five cells, 5-25Pm) inner
layer of smooth muscle cells (SMC) (facing submucosa) and the bulk of the circular muscle lamellae comprising 150-200 cells in the ileum (Figures 1-4). The inner layer often deviated slightly from the circular direction (Figures 1 and 2). This layer covered only the inner one-half to one-third of the tips (ridges) of the lamellae of circular muscle (Figure 1). The diameter of the innermost SMC tended to be smaller than the diameter of SMC of the outer circular layer (Figures 3 and 5). Interstitial cel1 types were intermingled with nerve fascicles of DMP; mast cells or eosinophils were only occasionally distinguishable (Figures 3 and 4). Electron Microscopy
of Deep Muscular Plexus
Smooth muscle cells. We could not discern between the two circular muscle layers with regard to filament constitution or contents of other organelles. In the electron microscope, there were no apparent differences in electron density between muscle cells of the two layers (Figures 5 and 7a). In both circular muscle layers, adherens junctions were present between SMC. Gap junctions were seen only between SMC of the outer circular layer (Figure 12a). Interstitial cells ofCaja1. In the inner region of the circular muscle layers, a cel1 type with several
January 1992
INTERSTITIAL
CELLS OF CAJAL IN THE DEEP MUSCULAR
PLEXUS
59
Fig ure 7. Interstitial cells of Cajal (*) in tangential section. Numerous caveolae lc, orrows) and a continuous hasal lamina are puesent. Gr.2mular endoplasmic reticulum and a Golgi apparatus (G) are apparent. A process of an outer circular muscle cel1 (OCM) CI ontacts the ICC (a, arrow; upper frame; and cl. A gap junction is formed with another process (ICC or SMC) (lower frame and inset). Ar1-owheads, cytoplasmic dense bodies. [Original magnifications: X14,600, bar = 1 urn (a); X83,800, bar = 0.1 urn (b, inset, lowel *frame in iI); and X46,000, bar = 0.5 pm (c, upper frame in a).]
60 RUMESSEN ET AL.
GASTROENTEROLOGY
Vol. 102, No. 1
Figure 8. Tangential section showing overlapping processes of ICC (*). A gap junction between processes is shown @ame and b, arrows). Arrowheads (b), cytoplasmic dense bodies. [Original magnifications: X5900, bar = 2 pm (a); X55,000, bar = 0.2pm (bl.1 Figure 9. (a) Interstitial cells of Cajal (*) penetrating the inner circular layer (ICM), a nerve fascicle is also located inside this layer (N). F, FLC; Su, submucosa; arrow, ICC processes ramifying in the inner circular layer. (Original magnification: X5200; bar = í! urn.) (bl Corresponding to frame in a. Interstitial cel1 of Cajal process and dense bodies are seen (arrowheads), and the cytoplasm is dominated by gER, intermediate filaments (smaZ1 arrows), and mitochondria. A basal lamina is clearly visible (braad arrows). Caveolae are inconspicuous (original magnification: X22,000; bar = 1 urn).
January 1992
INTERSTITIAL CELLS OF CAJAL IN THE DEEP MUSCULAR PLEXUS
61
Figure 10. (a) Interstitial cells of Cajal and overlapping processes (*) located at the submucosal border close to a submucous nerve fascicle (N). KM, inner circular layer. Frames correspond to b and c (original magnification X4600;bar = 4 pm). (b) Frame b from a. Two overlapping ICC processes; the cytoplasm is dominated hy intermediate filaments (lefi arrows) and dense bodies (arrowheads). Right arrow, coated vesicle. (Original magnification X30,400; bar = 0.5 pm.) (c) Detail of a (frame c) showing ICC cytoplasm dominated by gER (thick arrows), mitochondria (M), and smooth surfaced cisternae. Arrows, basal lamina; arrowheads, dense bands. (Original magnification ~44,300; bar = 0.5 pm.) Figure ll. (a) Same cells (*) as in Figure lOa, neighboring section. N, suhmucosal nerve fascicle; M, muscle cell. Frame, part b. (Original magnification %OttO; bar = 4 pm.) (b) Detail of a (frame) showing a submucosal nerve terminal (N) with clustering of vesicles and a presynaptic density (arrow) facing ICC cytoplasm. This is dominated by intermediate filaments and dense bodies (arrowheads), free ribosomes, cisternae of gER, and a large mitochondrion (M). Thick filaments are not apparent (original magnification X18,500; bar = 1 pm).
62 RUMESSEN ET AL.
GASTROENTEROLOGYVol. 102,No.l
Figure 12. Neighboring sections of the same area between the circular layers with details of ICC and nerves of DMP (a-h). N, nerve fascicle; OCM, outer circular layer; KM, inner circular layer; E, elastin fibrils; *, nuclei or processes of ICC. (a) Two ICC and several smal1 processes. Processes and cel1 bodies are interconnected with each other and with SMC by intermediate type junctions (arrowheads). Tbin arrows show patches of intermediate (10~nm) and thin (5nm) filaments (enlarged in inset, upper right). Cisternae of SER and gER are conspicuous. Thick arrow, gap junction between two muscle cells of the outer circular layer. [Original magnifications: x15,400, bar = 1 pm; x43,000, bar = 0.2pm (inset).] (b) Neighboring section; lipid droplets appear in ICC (L). Frames correspond to c and e (original magnification X15,400; bar = 1 pm). (c) Detail of b, showing close apposition of a nerve terminal to an ICC process. In the terminal, a presynaptic dense projection and clustering of vesicles is seen (arrow) (original magnification ~37,000; bar = 0.5 pm). (d) Neighboring section of c, showing several presynaptic densities closely apposed to ICC cytoplasm (between arrows). In the ICC, cisternae of SER are shown (arrowheads) (original magnification X37,000; bar = 0.5 pm). (e) Detail of b, showing ICC cytoplasm; SER and caveolae are shown (arrowheads and arrows) (original magnification x45,000; bar = 0.2 pm).
January
1992
INTERSTITIAL
CELLS
OF CAJAL IN THE DEEP MUSCULAR
PLEXUS
63
Figure 12 (cont’d.). (f ) Stil1 deeper in the tissue, several ICC processes are shown. M, muscle cells; large frame, shown in g; smal1 frame, shown in part i. (original magnification X10,000; bar = 2 urn.) (g) Large frame in part f. Dense bodies and dense bands are shown (arruws). Some dense bodies are surrounded by intermediate (lO-nm) and thin (8-nm) lilaments (circles) (Original magnification ~80,ooO; bar = 0.8 pm). (Ir)Stil1 deeper, the nucleated ICC shows several areas with dense bodies surrounded by intermediate (10~nm) and thin filaments (arrawheads). Dense bands and caveolae are not so common or regularly distributed as in smooth muscle cells (M). Note the close apposition of overlapping ICC processes. Arrow, synapselike arrangement with ICC process. (Original magnification x18,800; bar = 1 urn.) (i) Smallframe in f, showing ICC cytoplasm with thin (8nm) filaments (A) surrounding intermediate (10~nm) filaments (arrows). This organization very much resembles that of SMC (j), but thick (18nm) filaments are not seen. DB, dense band; BL, basal lamina. [Original magnification x149,000; bar = 0.1urn (100 nm).] (j) Area of SMC cytoplasm for comparison with i. Thick (1%nm) filaments (arrows) are conspicuous and surrounded by thin (8nm) filaments (A). DB, dense body. [Original magnification X149,000; bar = 0.1 urn (100 nm).]
64
RUMESSEN ET AL.
GASTROENTEROLOGY Vol. 102, No. 1
Figure 13. (o) An FLC with its nucleus (F) hetween the circular layers and a process piercing through the inner layer (ICM) ramifying in submucosa (Su). Frame is shown in b. SN, submucosal nerve; N, nerve fascicle of DMP. (Original magnification X5600; bar = 2 pm.) (b) Detail of a. The cytoplasm of the FLC is dominated by cisternae of gER (orrow). Note the absente of caveolae and a basal lamina. Mi, mitochondrion. (Original magnification X16,300; bar = 1 pm.) Figure 14. An MLC at the leve1 of DMP. Processes of an FLC are closely apposed to the cytoplasm of the MLC (orrows), which is dominated by primary lysosomes (arrowbead) and coated vesicles. N, nerve fascicle of DMP; M, smooth muscle cell; P, processes of the MLC. (Original magnitication X9200; bar = 2 pm.) Figure 15. An MLC inside the innermost layer of circular muscle (KM). c, collagen fihrils; Su, submucosa; arrowhead, primary lysosome. (Original magnification X9600; bar = 2 pm.)
INTERSTITIAL CELLS OF CAJAL IN THE DEEP MUSCLJLAR PLEXUS
January 1992
Tabje 1, A Survey of the Cytological Features of Interstitial the Smal1 Intestine in Different Mammals Species (Study)
Cells of Cajal Associated
With the Deep Muscular
65
Plexus in
Cav
BM
Su-SER
DB
gER
Mi
Gly
M
1
A
Sy
Gj-C
Gj-1
+ +
+ -
++ ++
+++ +
++ ++
+ +
+ -
+ -
+ +
+ +
+ -
+ ++
+ ++
+
+ t
-
-
-
-
++
0
t+
t+
+
0
+
+
+t
+t+ ttt
tt
Human Dog (6) Guinea pig (21) Mouse (8,9, 15, 20)
t
t to t+t, approximation of relative frequency; 0, absent; -, information not available. Cav, caveolae; BM, basal lamina; Su-SER, subsurface smooth endoplasmic reticulum; DB, dense bodies; gER, granular endoplasmic reticulum; Mi, mitochondria; Gly, glycogen; M, myosin (thick) filaments; 1, intermediate filaments; A, actin (thin) filaments; Sy, synapselike contacts with DMP; Gj-C, gap junctions with outer circular muscle cells; Gj-1, gap junctions with other ICC.
myoid features but clearly different from SMC, fibroblastlike cells (FLC), or other interstitial cel1 types was identified. According to our previously reported electron microscopical criteria,*-‘0~‘8 this cel1 type was recognized as ICC associated with DMP. Interstitial cells of Cajal were not clearly identifiable in routine toluidine blue-stained sections (Figures 3 and 4). However, the ultrastructural organization and the cytology clearly distinguished ICC from SMC and from FLC or other cellular elements associated with DMP. Compared with ICC associated with Auerbach’s plexus of the human smal1 intestine,” ICCDMP had a distinctly different and an even more myoid ultrastructure. ORGANIZATION
OF INTERSTITIAL
CELLS OF CA-
The ICC were not confined to the space between the two subdivisions of the circular muscle layer but were (less frequently) also ramifying in the inner zones of the outer division of the circular layer (Figure 5) as wel1 as inside the innermost layer (Figure 9a). Not seldom ICC were located at the submucosal border, sometimes close to submucosal nerve elements (Figures 10 and 11). Synapselike contacts with this plexus were suggested (Figure 11). Interstitial cells of Cajal were most often encountered as single cel1 profiles or smal1 bundles of overlapping, slender processes of two to three cells (Figures Ba and 12h) with apposition of large membrane areas separated by a rather constant 20-nm cleft. The processes of ICC were furthermore interconnected by intermediate (adherens) junctions and only very occasionally by smal1 gap junctions (Figures 7b and Bb). Reflexive (internal) gap junctions were also seen. The processes of ICC were chiefly bipolar in shape with their long axes oriented parallel to the long axes of SMC (Figures 7a and Ba). Two or more flattened processes very often extended in radial directions as wel1 (Figures 5 and 12a). Axons devoid of glial cel1 covering were often in close contact with ICC cytoplasm, and specializations of the presynaptic membrane (synapselike dense projections) were often apparent, together with a clustering of 50-nm clear, round vesicles (Figures lla and b and ZZc, d, and h). JAL.
Postjunctional specializations were not seen. Interstitial cells of Cajal established adherens-type junctions with circular muscle cells of both layers, but smal1 gap junctions between ICC and the outer circular muscle cells were encountered only occasionally. Some ICC contacted both circular layers by means of smal1 processes (Figure 6) or ball-andsocket connections (Figure 7a). The density of ICC at this leve1 of the smal1 intestine was comparable with that of FLC and appeared lower than the density of ICC between the main muscle layersl’ and lower than in mouse DMP.’ In contrast to mouse smal1 intestine,6~g~‘g there were no obvious specific relations between macrophagelike cells (MLC) and ICC; likewise, specialized contacts to FLC were lacking. CYTOLOGY OF INTERSTITIAL cELu 0F CAJAL. Because of the close similarity between the cytology of ICC and that of SMC, a correct identification necessitated a thorough inspection of neighboring grids and sections. This is illustrated in Figure 12a-j. These figures illustrate processes and the perinuclear cytoplasm of ICC between the circular muscle layers. The transition of an ICC profile with a cytology intermediate between FLC and SMC to a profile not easily distinguishable from an SMC is shown (compare Figure 12a and f). The basal lamina of ICC was continuous (Figures 7a and 9b). Caveolae, dense bands, and dense bodies were conspicuous although fewer in number compared with SMC (Figures 7a and lla). It was characteristic that the regularly repeated pattern of alternating dense bands and caveolae seen in SMC was disrupted in ICC (this is most evident in Figures 7a and 12a). Characteristic organelles included smooth endoplasmic reticulum (SER) (Figure 12a), intermediate filament bundles (Figures 9b and lob), abundant mitochondria, and sometimes lipid droplets (Figure 12b). The SER was characteristically distributed as subsurface cisternae and was most abundant in the smaller processes (Figure 12ce). The perinuclear cytoplasm of ICC was generally more sparse than the cytoplasm of SMC, and ICC nuclei were more irregular and contained more pe-
66 RLJMESSENET AL.
ripheral heterochromatin than SMC and FLC (Figure 9a). Some perinuclear areas were dominated by a relatively abundant flattened granular endoplasmic reticulum @ER) (Figures 7 and 10~1and c), whereas other areas of the same cells contained dense bodies and bundles of intermediate (lo-nm) filaments arranged in a very regular pattern parallel to thin (5nm, actin) filaments (Figure 12g-j). This organization very much resembled that of myosin (thick, 15 nm) filaments in SMC, but the diameter of the thickest filaments in ICC was clearly smaller, corresponding to 10 nm (intermediate filaments) (Figure 12i-j). Thus, thick filaments were not identified in ICC. Glycogen granules were inconspicuous irrespective of fixative used. Other constituents of ICC cytoplasm included free ribosomes and coated vesicles (which were inconspicuous in SMC), whereas lysosomes and Golgi areas were seldom encountered. Nervous elements and glial cells. Axons of DMP were generally less wel1 preserved than axons in the outer zones of the musculature. The best results were obtained with fixatives with the highest aldehyde concentrations (8% + 8% and 12% + 12% paraformaldehyde plus glutaraldehyde) and with picrate-supplemented fixative.” Nerve bundles contained 3-15 axons, ensheathed by typical glial cells (Figure 14). Based on the morphology of the vesicles, only two types of terminals were identified: (a) terminals containing a predominance of smal1 (50-nm), round agranular vesicles together with larger (100-nm) granular vesicles with a distinct halo (Figure 12c, d, and h); and (b) terminals with smal1 (50-nm), round agranular vesicles together with very large (150-200-nm) granular vesicles. Terminals containing smal1 dense core vesicles or a predominante of flattened vesicles or mitochondria were not encountered. Only terminals of the first type were in close, synapselike contact with ICC. Axons were only very occasionally in close contact with other cel1 types, including SMC. Nerve bundles piercing the outer circular layer were rather frequent, but, apart from DMP, no repeated pattern of organization was recognizable. Smal1 axon bundles sometimes pierced the innermost layer of SMC (Figure 9a). Glial cells had a thick, continuous basal lamina and contained abundant intermediate (10-nm) filaments. Caveolae, dense bodies, and dense bands were absent, and gER was scarce. Fibroblasthke cells and macrophagelike cells. Fibroblastlike cells were frequent between the circular layers as wel1 as inside the outer circular layer. They were easily distinguished from ICC, lacking basal lamina, caveolae, as wel1 as dense bands and dense bodies. Fibroblastlike cells did not form specialized contacts with other cells including SMC,
GASTROENTEROLOGYV~~.~~~,N~.I
and they were not in synapselike contact with axons. They had long slender (flattened) processes and an abundance of often moderately distended gER as wel1 as conspicuous intermediate filaments. Lysosomes and coated vesicles were frequently seen (Figure 13a and b). Occasionally FLC processes intermingled with and penetrated the innermost circular layer (Figure 13). A conspicuous cellular relation was the very intimate association of FLC processes and MLC (Figure 14). Macrophagelike cells were identified on the basis of our previously reported criteria” and had characteristic irregular contours, abundant primary lysosomes, coated vesicles, and coated pits. They were frequent in this region of the smal1 intestine and were sometimes encountered singly in the musculature, even inside the innermost circular layer (Figure 15). Intercellular space and vessels. Capillaries of the nonfenestrated type accompanied nerve fascicles of DMP. Collagen fibrils were rather abundant, also between individual SMC. Elastin fibrils were present (Figure 12h) but without obvious relations to ICC or other cel1 types. Discussion Ultrastructural studies of ICC associated with the DMP have been performed in mouse,8’Q’15*20 guinea pig,‘l dog,6 and humans.” The morphology and distribution of ICC-DMP has been investigated with zinc-iodide/osmic acid techniques and scanning electron microscopy.7*21~22 The ultrastructural features of ICC-DMP have many similarities in different species, but some important differences exist (Table 1). Human ICC-DMP resemble SMC more closely because of their filament organization and content of dense bodies. Thick (15-nm) filaments were not recognized in ICC-DMP, whereas they were readily identified in SMC. However, the presence or absente of myosin can only be determined by special techniques. The human ICC-DMP establish far fewer gap junctions with SMC or other ICC compared with other mammals, and the innervation of ICC seems more sparse compared with mouse smal1 intestine.* The cel1 contacts between ICC are primarily of the intermediate (adherens) type or consists of a large overlapping of processes. This organization resembles nodal tissue of the heart.23 In these respects the organization of ICC-DMP resembles that of ICC located at the leve1 of Auerbach’s plexus,” although the organization and the cytology differ considerably on several points. Interstitial cells of Cajal at the leve1 of Auerbach’s plexus are dominated by intermediate filaments, the basal lamina is discontinuous, and they form thicker bund1es.l’ We have been able to confirm some of the data
January 1992
INTERSTITIAL CELLS OF CATAL IN THE DEEP MUSCULAR PLEXUS
previously presented on human ICC-DMP.” As particularly emphasized in Figure 12, it may be problematic to correctly distinguish ICC-DMP from SMC. The frequency of ICC-DMP may therefore be underestimated.” The data presented here should suffice to identify most ICC processes. Furthermore, we have noticed that ICC-DMP are not confined to the connective tissue space between the subdivisions of the circular muscle but may extend for a variable distance into both circular layers as wel1 as bridge the two. In the present study, ICC-DMP were found at the submucosal border; this has not been described before in any species. These relationships may be important for the interpretation of data concerning the coupling of the circular layers and a possible influence of both submucosal nerves and axons from Auerbach’s plexus on the circular muscle. The functional and immunochemical significante of the close association between ICC and the so-called synapselike specializations has yet to be clarified. Further studies are also necessary to test whether ICC located at the leve1 of Auerbach’s plexus are coupled to ICC-DMP and whether ICC-DMP may be involved in the coupling of the parallel lamellae of circular muscle, which are very wel1 developed in humans. A corresponding plexus of ICC located at the submucosal border of the external colonic musculature has been described in several speciesz4-” Electrophysiological evidente from canine colon suggests that these cells are colonic pacemaker cells.28*2gThey are innervated by vasoactive intestinal polypeptidecontaining nerves and may mediate inhibitory input to the colonic circular muscle.30-32 Similar studies on the smal1 intestine are lacking. Vasoactive intestinal polypeptide is preferentially located in the DMP in canine ileum,” and, in analogy with the findings in canine colon, it is possible that DMP exerts inhibitory input to the circular muscle through innervation of ICC. Electrophysiological studies on this region of the smal1 boweP4 have suggested that nonneural cells located between the main muscle layers and between the outer and inner subdivisions of the circular layer generate slow waves in several species, including humans. Although studies on human smal1 intestine are preliminary,14 it is therefore also possible that ICC-DMP may act in concert with ICC located at the leve1 of Auerbach’s plexus as smal1 intestinal pacemaker ce11s.gJo,33*34 It has been speculated that the region between the two circular layers would be an optimal position for external muscular mechanoreceptors.5s6**J6 The ultrastructure of mechanosensitive neurons and target cells in the circular muscle is unknown, but terminals of intrinsic afferent neurons may contact ICC both at the levels of Auerbach’s plexus and in the DMP. Vagal tension receptors are located in the ex-
67
ternal muscle layers,35 but the morphology and the distribution of these nerves and their target cells is also unknown. The morphology and the distribution of ICC fits wel1 with physiological concepts of tension receptor localization and shape.8,‘6*3” In conclusion, the present study has provided an ultrastructural description of a separate myoid cel1 type located in the innermost zone of the external muscle of the human smal1 intestine. These cells correspond to ICC-DMP of other mammals, and although the organization appears more diffuse, the cytology and organization of ICC-DMP is compatible with regulatory functions also in human smal1 intestine. The present study provides a basis for the interpretation of future physiological and pathological studies of this area of the human smal1 intestine. References 1. Cajal SR. Histologie
du système nerveux de 1’Homme et des Vertébrés. Paris: Maloine, 1911. Über die Struktur des Zir2. Li PL. Neue Beobachtungen kulärmuskels in Dünndarm bei Wirbeltieren. Eine vergleichende Studie. Z Anat Entw Gesch 1937;107:212-222. 3. Li PL. The intramural nervous system of the smal1 intestine with special reference to the innervation of the inner subdivision of its circular muscle. J Anat 1940;74:34a-359. 4. Gabella G. Innervation of the intestinal muscular coat. J Neurocytol 1972;1:341-362. 5. Gabella G. Special muscle cells and their innervation in the smal1 intestine. Cel1 Tissue Res 1974;153:63-77. 6. Duchon G, Henderson R, Daniel EE. Circular muscle layers in the smal1 intestine. In: Daniel EE, ed. Proceedings of the 4th International Symposium of Gastrointestinal Motility, Banff, Alberta, Canada: Mitchell, 1974:635-646. 7. Rumessen JJ, Thuneberg L. Plexus muscularis profundus and associated interstitial cells. 1. Light microscopical studies of mouse smal1 intestine. Anat Rec 1982;203:115-127. 8. Rumessen JJ, Thuneberg L, Mikkelsen HB. Plexus muscularis profundus and associated interstitial cells. 11.Ultrastructural studies of mouse smal1 intestine. Anat Rec 1982;203:129-146. 9. Thuneberg L. Interstitial cells of Cajal: intestinal pacemaker cells? Adv Anat Embryo1 Cel1 Bio1 1982;71:1-130. 10. Thuneberg L. Interstitial cells of Cajal. In: Wood JD, ed. Handbook of physiology, the gastrointestinal system. Volume 1. Bethesda, MD: American Physiological Society, 1989:349-386. ll. Berezin 1, Sheppard S, Daniel EE, Yanaihara N. Ultrastructural immunocytochemical distribution of VIP-like immunoreactivity in dog ileum. Regul Pept 1985;11:287-298. 12. Wilson AJ, Llewellyn-Smith IJ, Furness JB, Costa M. The source of nerve fibers forming the deep muscular and circular muscle plexuses in the smal1 intestine of the guinea pig. Cel1 Tissue Res 1987;247:497-504. 13. Llewellyn-Smith IJ, Furness JB, Gibbins IL, Costa M. Quantitative ultrastructural analysis of enkephalin-substance P-, and VIP-immunoreactive nerve fibers in the circular muscle of the guinea pig smal1 intestine. J Comp Neurol 1988; 272:139-148. 14. Hara Y, Kubota M, Szurszewski JH. Electrophysiology of smooth muscle of the smal1 intestine of some mammals. J Physiol 1986;372:501-520. 15. Yamamoto M. Electron microscopic studies on the innervation of the smooth muscle and the interstitial cel1 of Calal in
68
RUMESSEN ET AL.
the smal1 intestine of the mouse and bat. Arch Histol Jpn 1977;40:171-201. 16. Daniel EE. Nerves and motor activity of the gut. In: Brooks FP, Evers PW, eds. Nerves and the gut, Thorofare, NJ: Slack,
GASTROENTEROLOGY Vol. 102. No. 1
28. Barajos-Lopez
29.
1977:154-196. 17. Faussone-Pellegrini
18.
19.
20.
21.
22.
23.
24.
25.
26.
27.
MS, Cortesini C. Some ultrastructural features of the muscular coat of the human smal1 intestine. Acts Anat 1983;115:47-68. Rumessen JJ, Thuneberg L. Interstitial cells of Cajal in human smal1 intestine. Ultrastructural identification and organization between the main smooth muscle layers. Gastroenterology 1991;100:1417-1431. Mikkelsen HB, Thuneberg L, Rumessen JJ, Thorball N. Macrophage-like cells in the muscularis externa of the mouse smal1 intestine. Anat Rec 1985;213:77-86. Faussone-Pellegrini MS. Morphogenesis of the special circular muscle layer and of the interstitial cells of Cajal related to the plexus muscularis profundus of mouse intestinal muscle coat. An EM study. Anat Embryo1 1984;169:151-158. Kobayashi S, Suzuki M, Nishisaka T. Severed nerve stumps around a laser-irradiated lotus in the deep muscular plexus of the guinea-pig smal1 intestine. Arch Histol Cytol 1989; 52(Suppl):147-159. Kobayashi S, Furness JB, Smith T, Pompolo S. Histological identification of the interstitial cells of Cajal in the guinea-pig smal1 intestine. Arch Histol Jpn 1989;52:267-286. Tranum-Jensen J. The fine structure of the sinus node: a survey. In: Bonk FIM, ed. The sinus node. Structure, function and clinical relevante. Dordrecht, The Netherlands: Nijhoff, 1978:149-165. Stach W. Der Plexus Entericus Extremus des Dickdarmes und Seine Beziehungen zu den Interstitiellen Zellen (Cajal). Z Mikrosk Anat Forsch 1972;85:245-272. Faussone-Pellegrini MS. Ultrastructural peculiarities of the inner portion of the circular layer of the colon. Research in the mouse. Acts Anat 1985;122:187-192. Christensen J, Riek GA. Intrinsic nerves in the mammalian colon: confirmation of a plexus at the circular muscle-submucosal interface. J Auton Nerv Syst 1987;21:223-231. Berezin 1, Huizinga JD, Daniel EE. Interstitial cells of Cajal in the canine colon: a special communication network at the inner border of the circular muscle. J Comp Neurol 1988;273:42-51.
30.
31.
32.
33.
34.
35.
36.
C, Berezin 1, Daniel EE, Huizinga JD. Pacemaker activity recorded in interstitial cells of the gastrointestina1 tract. Am J Physiol 1989;257:C830-C835. Langton P, Ward SM, Carl A, Norell MA, Sanders KM. Spontaneous electrical activity of interstitial cells Cajal isolated from canine proximal colon. Proc Nat1 Acad Sci USA 1989;86:7280-7284, Berezin 1, Huizinga JD, Farraway L, Daniel EE. Innervation of interstial cells of Cajal by vasoactive intestinal polypeptide containing nerves in canine colon. Can J Physiol Pharmacol 1990;68:922-932. Smith TK, Reed JB, Sanders KM. Electrical pacemakers of canine proximal colon are functionally innervated by inhibitory motor neurons. Am J Physiol 1989;252:C466-C477. Huizinga JD, Berezin 1, Daniel EE, Chow E. Inhibitory innervation of colonic smooth muscle cells and interstitial cells of Cajal. Can J Physiol Pharmacol 1990;68:447-454. 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. Thuneberg L, Johansen V, Rumessen JJ, Andersen BG. Interstitial cells of Cajal: selective uptake of methylene blue inhibits slow wave activity. In: Roman C, ed. Gastrointestinal motility. Lancaster, England: MTP, 1984,495-502. Iggo A. Gastro-intestinal tension receptors with unmyelinated afferent fibres in the vagus of the cat. Q J Exp Physiol 1957;42:130-143. Leek BF. Abdominal visceral receptors. In: Neil E, ed. Handbook of sensory physiology. Volume 3. New York: Springer, 1972:113-129.
Received November 8, 1990. Accepted April 23,199l. Address requests for reprints to: Dr. Jiiri Johannes Rumessen, Department of Anatomy C, University of Copenhagen, Panum Instituttet, Blegdamsvej 3, DK-2200 Copenhagen, Denmark. Supported by The Novo Foundation and Holger Hjortenbergs Fond. The authors thank S. Peters, K. Stub-Christensen, and V. Heidemann for technical assistance. The helpful assistance of the staff of Department of Surgery D, Gentofte Hospital, and of Department of Surgery C, Rigshospitalet, is gratefully acknowledged.
1992:102:56-68
Ultrastructure of Interstitial Cells of Cajal Associated With Deep Muscular Plexus of Human Smal1 Intestine JÜRI JOHANNES RUMESSEN, and LARS THUNEBERG
HANNE
BIRTE
MIKKELSEN,
Department of Anatomy C, University of Copenhagen, Panum Instituttet, Copenhagen, Denmark
Evidente showing that interstitial cells of Cajal have important regulatory functions in the gut musculature is accumulating. In the current study, the ultrastructure of the deep muscular plexus and associated interstial cells of Cajal in human smal1 intestine were studied to provide a reference for identification and further physiological or pathological studies. The deep muscular plexus was sandwiched between a thin inner layer of smooth muscle (one to five cells thick) and the bulk of the circular muscle. Interstitial cells of Cajal in this region very much resembled smooth muscle cells (with a continuous basal lamina, caveolae, intermediate filaments, dense bodies, dense bands, and a well-developed subsurface smooth endoplasmic reticulum), but the arrangement of organelles was clearly different, and cisternae of granular endoplasmic reticulum were abundant. Interstitial cells of Cajal were distinguished from fibroblasts or macrophages in the region. They ramified in the inner zone of the outer division of circular muscle, penetrated the innermost circular layer, and were also found at the submucosal border. They were in close, synapselike contact with nerve terminals of the deep muscular plexus, and only few gap junctions with other interstitial cells of Cajal or with the musculature were observed. Compared with interstitial cells of Cajal from other mammals, those associated with the deep muscular plexus in the human smal1 intestine more closely resemble smooth muscle cells, and their organization appears more diffuse; however, the ultrastructure and organization of interstitial cells of Cajal is compatible with modulatory actions on the circular muscle also in humans.
1
n the smal1 intestinal muscularis externa, the deep muscular plexus (DMP or plexus muscularis profundus) occupies a narrow space between the thick outer and thin inner layer of circular muscle cells.l-‘o The potential importante of DMP as an intrinsic neural regulator of circular muscle function is becoming increasingly clear.“-‘3 Slow waves of the
smal1 intestine may, in part, originate in nonneural cells located between the two subdivisions of the circular muscle layer.14 The exact cellular origin has not been established. Ultrastructural studies of this region in different animal species have focused on the importante of interstitial cells of Cajal (ICC) as myoid cells intercalated between the DMP and the circular musc1e.6-g,‘5*‘6Based on this, a key role of ICC-DMP as regulators of circular muscle function has been strongly suggested. The ultrastructure of DMP and associated ICC in human smal1 intestine has been studied very little.17 In the present study we extend our previous observations of ICC in mouse DMP7-’ to human smal1 intestine, with emphasis on a clear identification of ICC and their detailed ultrastructural organization. In conjunction with our recent study of ICC in the region between the main muscle layers,18 the present results may provide a framework for future electrophysiological as wel1 as pathologica1 studies of human smal1 intestine. Materials and Methods Samples of freshly resected, uninvolved smal1 intestine (2 from the duodenum, 2 from the jejunum, and 10 from the ileum) were obtained from 12 adult patients (3 women and 9 men, aged 27-90 years; median, 60 years) undergoing surgery because of incarceration of inguinal hernia or gastric, pancreatic, or colonic malignancies. The patients had no other gastrointestinal disease, and patients with peritoneal carcinosis were excluded. The tissue was handled and processed for transmission electronmicroscopy (Philips EM 300 and 400; Eindhoven, The Netherlands) as previously described.” After resection, l-2-cm pieces of smal1 intestine were immediately cut and immersed in the following two fixatives: (a) a modified Karnovsky fixative” containing 2% + 2%, 8% + 8%, or 12% + 12% glutaraldehyde (Taab, Berkshire, England) and paraformaldehyde (Merck, Darmstadt, Germany) in 0.1 mol/L phosphate buffer (Merck), pH 7.3, at 20°C for at 0 1992 by the American Gastroenterological 0016-5085/92/$3.00
Association
January 1992
INTERSTITIAL CELLS OF CAJAL IN THE DEEP MUSCULAR
PLEXUS
57
Figure 1. Circular muscle lamina in cross-section. Deep muscular plexus is distinctly located between tbe thin innermost layer of circular muscle cells (arrows) and the outer bulk of circular muscle (OCM) but fades out towards Auerbach’s plexus (arrowheads). S, septa between circular muscle laminae; SU, submucosa. (Toluidine blue; original magnification X260; bar = 50 pm.) Figure 2. Innermost zone of the circular muscle. The innermost layer (arrows) deviates (OCM) (toluidine blue, original magnification X400; bar = 50 pm).
from the circular
direction
of the outer layer
Figures 3 and 4. Cross-section (Figure 3) and tangential (Figure 4) section of DMP and associated interstitial elements as they appear in routine light microscopical sections stained with toluidine blue. Identification of different cel1 types labeled here requires electron microscopy at higher magnifications (sec later figures). Arrow, ICC; F, FLC; M, MLC; S, glial cell; MC, mast cell; N, nerve fascicle; C, capillary; Su, submucosa; OCM, outer circular layer; KM, inner circular layer. [Original magnifications: X960, bar = 20 pm (Figure 3); X1050, bar = 20 pm (Figure 4).]
58
RUMESSEN ET AL.
GASTROENTEROLOGY Vol. 102, No. 1
Figure 5. Interstitial cells of Cajal (*) ramifying in the inner zone of the outer circular layer (M). ICM, inner circular layer. (Original magnification X9999; bar = 2 pm.) Figure 6. Interstitial cells of Cajal (*) contacting two muscle cells and bridging the two circular layers. OCM, outer circular layer; ICM, inner circular layer. (Original magnification X17,800; bar = 1 Pm.1
least z hours; and (b) a modified Bouin 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 0.18%, 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% OsO, in 0.1 mol/ L phosphate buffer for 2 hours. The pieces were dehydrated in alcohol, black-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, and ultrathin (70 nm) sections were cut, mounted on copper grids, and contrasted with uranyl acetate and lead citrate. The study was performed in accordance with the Helsinki Declaration 11 and approved by the ethica1 committees of Copenhagen and Copenhagen County.
Results We found no differences in the principal arrangement of ICC and DMP in the different parts of
the smal1 intestine. Light Microscopy
of Deep Muscular Plexus
The deep muscular plexus was sandwiched between a thin (one to five cells, 5-25Pm) inner
layer of smooth muscle cells (SMC) (facing submucosa) and the bulk of the circular muscle lamellae comprising 150-200 cells in the ileum (Figures 1-4). The inner layer often deviated slightly from the circular direction (Figures 1 and 2). This layer covered only the inner one-half to one-third of the tips (ridges) of the lamellae of circular muscle (Figure 1). The diameter of the innermost SMC tended to be smaller than the diameter of SMC of the outer circular layer (Figures 3 and 5). Interstitial cel1 types were intermingled with nerve fascicles of DMP; mast cells or eosinophils were only occasionally distinguishable (Figures 3 and 4). Electron Microscopy
of Deep Muscular Plexus
Smooth muscle cells. We could not discern between the two circular muscle layers with regard to filament constitution or contents of other organelles. In the electron microscope, there were no apparent differences in electron density between muscle cells of the two layers (Figures 5 and 7a). In both circular muscle layers, adherens junctions were present between SMC. Gap junctions were seen only between SMC of the outer circular layer (Figure 12a). Interstitial cells ofCaja1. In the inner region of the circular muscle layers, a cel1 type with several
January 1992
INTERSTITIAL
CELLS OF CAJAL IN THE DEEP MUSCULAR
PLEXUS
59
Fig ure 7. Interstitial cells of Cajal (*) in tangential section. Numerous caveolae lc, orrows) and a continuous hasal lamina are puesent. Gr.2mular endoplasmic reticulum and a Golgi apparatus (G) are apparent. A process of an outer circular muscle cel1 (OCM) CI ontacts the ICC (a, arrow; upper frame; and cl. A gap junction is formed with another process (ICC or SMC) (lower frame and inset). Ar1-owheads, cytoplasmic dense bodies. [Original magnifications: X14,600, bar = 1 urn (a); X83,800, bar = 0.1 urn (b, inset, lowel *frame in iI); and X46,000, bar = 0.5 pm (c, upper frame in a).]
60 RUMESSEN ET AL.
GASTROENTEROLOGY
Vol. 102, No. 1
Figure 8. Tangential section showing overlapping processes of ICC (*). A gap junction between processes is shown @ame and b, arrows). Arrowheads (b), cytoplasmic dense bodies. [Original magnifications: X5900, bar = 2 pm (a); X55,000, bar = 0.2pm (bl.1 Figure 9. (a) Interstitial cells of Cajal (*) penetrating the inner circular layer (ICM), a nerve fascicle is also located inside this layer (N). F, FLC; Su, submucosa; arrow, ICC processes ramifying in the inner circular layer. (Original magnification: X5200; bar = í! urn.) (bl Corresponding to frame in a. Interstitial cel1 of Cajal process and dense bodies are seen (arrowheads), and the cytoplasm is dominated by gER, intermediate filaments (smaZ1 arrows), and mitochondria. A basal lamina is clearly visible (braad arrows). Caveolae are inconspicuous (original magnification: X22,000; bar = 1 urn).
January 1992
INTERSTITIAL CELLS OF CAJAL IN THE DEEP MUSCULAR PLEXUS
61
Figure 10. (a) Interstitial cells of Cajal and overlapping processes (*) located at the submucosal border close to a submucous nerve fascicle (N). KM, inner circular layer. Frames correspond to b and c (original magnification X4600;bar = 4 pm). (b) Frame b from a. Two overlapping ICC processes; the cytoplasm is dominated hy intermediate filaments (lefi arrows) and dense bodies (arrowheads). Right arrow, coated vesicle. (Original magnification X30,400; bar = 0.5 pm.) (c) Detail of a (frame c) showing ICC cytoplasm dominated by gER (thick arrows), mitochondria (M), and smooth surfaced cisternae. Arrows, basal lamina; arrowheads, dense bands. (Original magnification ~44,300; bar = 0.5 pm.) Figure ll. (a) Same cells (*) as in Figure lOa, neighboring section. N, suhmucosal nerve fascicle; M, muscle cell. Frame, part b. (Original magnification %OttO; bar = 4 pm.) (b) Detail of a (frame) showing a submucosal nerve terminal (N) with clustering of vesicles and a presynaptic density (arrow) facing ICC cytoplasm. This is dominated by intermediate filaments and dense bodies (arrowheads), free ribosomes, cisternae of gER, and a large mitochondrion (M). Thick filaments are not apparent (original magnification X18,500; bar = 1 pm).
62 RUMESSEN ET AL.
GASTROENTEROLOGYVol. 102,No.l
Figure 12. Neighboring sections of the same area between the circular layers with details of ICC and nerves of DMP (a-h). N, nerve fascicle; OCM, outer circular layer; KM, inner circular layer; E, elastin fibrils; *, nuclei or processes of ICC. (a) Two ICC and several smal1 processes. Processes and cel1 bodies are interconnected with each other and with SMC by intermediate type junctions (arrowheads). Tbin arrows show patches of intermediate (10~nm) and thin (5nm) filaments (enlarged in inset, upper right). Cisternae of SER and gER are conspicuous. Thick arrow, gap junction between two muscle cells of the outer circular layer. [Original magnifications: x15,400, bar = 1 pm; x43,000, bar = 0.2pm (inset).] (b) Neighboring section; lipid droplets appear in ICC (L). Frames correspond to c and e (original magnification X15,400; bar = 1 pm). (c) Detail of b, showing close apposition of a nerve terminal to an ICC process. In the terminal, a presynaptic dense projection and clustering of vesicles is seen (arrow) (original magnification ~37,000; bar = 0.5 pm). (d) Neighboring section of c, showing several presynaptic densities closely apposed to ICC cytoplasm (between arrows). In the ICC, cisternae of SER are shown (arrowheads) (original magnification X37,000; bar = 0.5 pm). (e) Detail of b, showing ICC cytoplasm; SER and caveolae are shown (arrowheads and arrows) (original magnification x45,000; bar = 0.2 pm).
January
1992
INTERSTITIAL
CELLS
OF CAJAL IN THE DEEP MUSCULAR
PLEXUS
63
Figure 12 (cont’d.). (f ) Stil1 deeper in the tissue, several ICC processes are shown. M, muscle cells; large frame, shown in g; smal1 frame, shown in part i. (original magnification X10,000; bar = 2 urn.) (g) Large frame in part f. Dense bodies and dense bands are shown (arruws). Some dense bodies are surrounded by intermediate (lO-nm) and thin (8-nm) lilaments (circles) (Original magnification ~80,ooO; bar = 0.8 pm). (Ir)Stil1 deeper, the nucleated ICC shows several areas with dense bodies surrounded by intermediate (10~nm) and thin filaments (arrawheads). Dense bands and caveolae are not so common or regularly distributed as in smooth muscle cells (M). Note the close apposition of overlapping ICC processes. Arrow, synapselike arrangement with ICC process. (Original magnification x18,800; bar = 1 urn.) (i) Smallframe in f, showing ICC cytoplasm with thin (8nm) filaments (A) surrounding intermediate (10~nm) filaments (arrows). This organization very much resembles that of SMC (j), but thick (18nm) filaments are not seen. DB, dense band; BL, basal lamina. [Original magnification x149,000; bar = 0.1urn (100 nm).] (j) Area of SMC cytoplasm for comparison with i. Thick (1%nm) filaments (arrows) are conspicuous and surrounded by thin (8nm) filaments (A). DB, dense body. [Original magnification X149,000; bar = 0.1 urn (100 nm).]
64
RUMESSEN ET AL.
GASTROENTEROLOGY Vol. 102, No. 1
Figure 13. (o) An FLC with its nucleus (F) hetween the circular layers and a process piercing through the inner layer (ICM) ramifying in submucosa (Su). Frame is shown in b. SN, submucosal nerve; N, nerve fascicle of DMP. (Original magnification X5600; bar = 2 pm.) (b) Detail of a. The cytoplasm of the FLC is dominated by cisternae of gER (orrow). Note the absente of caveolae and a basal lamina. Mi, mitochondrion. (Original magnification X16,300; bar = 1 pm.) Figure 14. An MLC at the leve1 of DMP. Processes of an FLC are closely apposed to the cytoplasm of the MLC (orrows), which is dominated by primary lysosomes (arrowbead) and coated vesicles. N, nerve fascicle of DMP; M, smooth muscle cell; P, processes of the MLC. (Original magnitication X9200; bar = 2 pm.) Figure 15. An MLC inside the innermost layer of circular muscle (KM). c, collagen fihrils; Su, submucosa; arrowhead, primary lysosome. (Original magnification X9600; bar = 2 pm.)
INTERSTITIAL CELLS OF CAJAL IN THE DEEP MUSCLJLAR PLEXUS
January 1992
Tabje 1, A Survey of the Cytological Features of Interstitial the Smal1 Intestine in Different Mammals Species (Study)
Cells of Cajal Associated
With the Deep Muscular
65
Plexus in
Cav
BM
Su-SER
DB
gER
Mi
Gly
M
1
A
Sy
Gj-C
Gj-1
+ +
+ -
++ ++
+++ +
++ ++
+ +
+ -
+ -
+ +
+ +
+ -
+ ++
+ ++
+
+ t
-
-
-
-
++
0
t+
t+
+
0
+
+
+t
+t+ ttt
tt
Human Dog (6) Guinea pig (21) Mouse (8,9, 15, 20)
t
t to t+t, approximation of relative frequency; 0, absent; -, information not available. Cav, caveolae; BM, basal lamina; Su-SER, subsurface smooth endoplasmic reticulum; DB, dense bodies; gER, granular endoplasmic reticulum; Mi, mitochondria; Gly, glycogen; M, myosin (thick) filaments; 1, intermediate filaments; A, actin (thin) filaments; Sy, synapselike contacts with DMP; Gj-C, gap junctions with outer circular muscle cells; Gj-1, gap junctions with other ICC.
myoid features but clearly different from SMC, fibroblastlike cells (FLC), or other interstitial cel1 types was identified. According to our previously reported electron microscopical criteria,*-‘0~‘8 this cel1 type was recognized as ICC associated with DMP. Interstitial cells of Cajal were not clearly identifiable in routine toluidine blue-stained sections (Figures 3 and 4). However, the ultrastructural organization and the cytology clearly distinguished ICC from SMC and from FLC or other cellular elements associated with DMP. Compared with ICC associated with Auerbach’s plexus of the human smal1 intestine,” ICCDMP had a distinctly different and an even more myoid ultrastructure. ORGANIZATION
OF INTERSTITIAL
CELLS OF CA-
The ICC were not confined to the space between the two subdivisions of the circular muscle layer but were (less frequently) also ramifying in the inner zones of the outer division of the circular layer (Figure 5) as wel1 as inside the innermost layer (Figure 9a). Not seldom ICC were located at the submucosal border, sometimes close to submucosal nerve elements (Figures 10 and 11). Synapselike contacts with this plexus were suggested (Figure 11). Interstitial cells of Cajal were most often encountered as single cel1 profiles or smal1 bundles of overlapping, slender processes of two to three cells (Figures Ba and 12h) with apposition of large membrane areas separated by a rather constant 20-nm cleft. The processes of ICC were furthermore interconnected by intermediate (adherens) junctions and only very occasionally by smal1 gap junctions (Figures 7b and Bb). Reflexive (internal) gap junctions were also seen. The processes of ICC were chiefly bipolar in shape with their long axes oriented parallel to the long axes of SMC (Figures 7a and Ba). Two or more flattened processes very often extended in radial directions as wel1 (Figures 5 and 12a). Axons devoid of glial cel1 covering were often in close contact with ICC cytoplasm, and specializations of the presynaptic membrane (synapselike dense projections) were often apparent, together with a clustering of 50-nm clear, round vesicles (Figures lla and b and ZZc, d, and h). JAL.
Postjunctional specializations were not seen. Interstitial cells of Cajal established adherens-type junctions with circular muscle cells of both layers, but smal1 gap junctions between ICC and the outer circular muscle cells were encountered only occasionally. Some ICC contacted both circular layers by means of smal1 processes (Figure 6) or ball-andsocket connections (Figure 7a). The density of ICC at this leve1 of the smal1 intestine was comparable with that of FLC and appeared lower than the density of ICC between the main muscle layersl’ and lower than in mouse DMP.’ In contrast to mouse smal1 intestine,6~g~‘g there were no obvious specific relations between macrophagelike cells (MLC) and ICC; likewise, specialized contacts to FLC were lacking. CYTOLOGY OF INTERSTITIAL cELu 0F CAJAL. Because of the close similarity between the cytology of ICC and that of SMC, a correct identification necessitated a thorough inspection of neighboring grids and sections. This is illustrated in Figure 12a-j. These figures illustrate processes and the perinuclear cytoplasm of ICC between the circular muscle layers. The transition of an ICC profile with a cytology intermediate between FLC and SMC to a profile not easily distinguishable from an SMC is shown (compare Figure 12a and f). The basal lamina of ICC was continuous (Figures 7a and 9b). Caveolae, dense bands, and dense bodies were conspicuous although fewer in number compared with SMC (Figures 7a and lla). It was characteristic that the regularly repeated pattern of alternating dense bands and caveolae seen in SMC was disrupted in ICC (this is most evident in Figures 7a and 12a). Characteristic organelles included smooth endoplasmic reticulum (SER) (Figure 12a), intermediate filament bundles (Figures 9b and lob), abundant mitochondria, and sometimes lipid droplets (Figure 12b). The SER was characteristically distributed as subsurface cisternae and was most abundant in the smaller processes (Figure 12ce). The perinuclear cytoplasm of ICC was generally more sparse than the cytoplasm of SMC, and ICC nuclei were more irregular and contained more pe-
66 RLJMESSENET AL.
ripheral heterochromatin than SMC and FLC (Figure 9a). Some perinuclear areas were dominated by a relatively abundant flattened granular endoplasmic reticulum @ER) (Figures 7 and 10~1and c), whereas other areas of the same cells contained dense bodies and bundles of intermediate (lo-nm) filaments arranged in a very regular pattern parallel to thin (5nm, actin) filaments (Figure 12g-j). This organization very much resembled that of myosin (thick, 15 nm) filaments in SMC, but the diameter of the thickest filaments in ICC was clearly smaller, corresponding to 10 nm (intermediate filaments) (Figure 12i-j). Thus, thick filaments were not identified in ICC. Glycogen granules were inconspicuous irrespective of fixative used. Other constituents of ICC cytoplasm included free ribosomes and coated vesicles (which were inconspicuous in SMC), whereas lysosomes and Golgi areas were seldom encountered. Nervous elements and glial cells. Axons of DMP were generally less wel1 preserved than axons in the outer zones of the musculature. The best results were obtained with fixatives with the highest aldehyde concentrations (8% + 8% and 12% + 12% paraformaldehyde plus glutaraldehyde) and with picrate-supplemented fixative.” Nerve bundles contained 3-15 axons, ensheathed by typical glial cells (Figure 14). Based on the morphology of the vesicles, only two types of terminals were identified: (a) terminals containing a predominance of smal1 (50-nm), round agranular vesicles together with larger (100-nm) granular vesicles with a distinct halo (Figure 12c, d, and h); and (b) terminals with smal1 (50-nm), round agranular vesicles together with very large (150-200-nm) granular vesicles. Terminals containing smal1 dense core vesicles or a predominante of flattened vesicles or mitochondria were not encountered. Only terminals of the first type were in close, synapselike contact with ICC. Axons were only very occasionally in close contact with other cel1 types, including SMC. Nerve bundles piercing the outer circular layer were rather frequent, but, apart from DMP, no repeated pattern of organization was recognizable. Smal1 axon bundles sometimes pierced the innermost layer of SMC (Figure 9a). Glial cells had a thick, continuous basal lamina and contained abundant intermediate (10-nm) filaments. Caveolae, dense bodies, and dense bands were absent, and gER was scarce. Fibroblasthke cells and macrophagelike cells. Fibroblastlike cells were frequent between the circular layers as wel1 as inside the outer circular layer. They were easily distinguished from ICC, lacking basal lamina, caveolae, as wel1 as dense bands and dense bodies. Fibroblastlike cells did not form specialized contacts with other cells including SMC,
GASTROENTEROLOGYV~~.~~~,N~.I
and they were not in synapselike contact with axons. They had long slender (flattened) processes and an abundance of often moderately distended gER as wel1 as conspicuous intermediate filaments. Lysosomes and coated vesicles were frequently seen (Figure 13a and b). Occasionally FLC processes intermingled with and penetrated the innermost circular layer (Figure 13). A conspicuous cellular relation was the very intimate association of FLC processes and MLC (Figure 14). Macrophagelike cells were identified on the basis of our previously reported criteria” and had characteristic irregular contours, abundant primary lysosomes, coated vesicles, and coated pits. They were frequent in this region of the smal1 intestine and were sometimes encountered singly in the musculature, even inside the innermost circular layer (Figure 15). Intercellular space and vessels. Capillaries of the nonfenestrated type accompanied nerve fascicles of DMP. Collagen fibrils were rather abundant, also between individual SMC. Elastin fibrils were present (Figure 12h) but without obvious relations to ICC or other cel1 types. Discussion Ultrastructural studies of ICC associated with the DMP have been performed in mouse,8’Q’15*20 guinea pig,‘l dog,6 and humans.” The morphology and distribution of ICC-DMP has been investigated with zinc-iodide/osmic acid techniques and scanning electron microscopy.7*21~22 The ultrastructural features of ICC-DMP have many similarities in different species, but some important differences exist (Table 1). Human ICC-DMP resemble SMC more closely because of their filament organization and content of dense bodies. Thick (15-nm) filaments were not recognized in ICC-DMP, whereas they were readily identified in SMC. However, the presence or absente of myosin can only be determined by special techniques. The human ICC-DMP establish far fewer gap junctions with SMC or other ICC compared with other mammals, and the innervation of ICC seems more sparse compared with mouse smal1 intestine.* The cel1 contacts between ICC are primarily of the intermediate (adherens) type or consists of a large overlapping of processes. This organization resembles nodal tissue of the heart.23 In these respects the organization of ICC-DMP resembles that of ICC located at the leve1 of Auerbach’s plexus,” although the organization and the cytology differ considerably on several points. Interstitial cells of Cajal at the leve1 of Auerbach’s plexus are dominated by intermediate filaments, the basal lamina is discontinuous, and they form thicker bund1es.l’ We have been able to confirm some of the data
January 1992
INTERSTITIAL CELLS OF CATAL IN THE DEEP MUSCULAR PLEXUS
previously presented on human ICC-DMP.” As particularly emphasized in Figure 12, it may be problematic to correctly distinguish ICC-DMP from SMC. The frequency of ICC-DMP may therefore be underestimated.” The data presented here should suffice to identify most ICC processes. Furthermore, we have noticed that ICC-DMP are not confined to the connective tissue space between the subdivisions of the circular muscle but may extend for a variable distance into both circular layers as wel1 as bridge the two. In the present study, ICC-DMP were found at the submucosal border; this has not been described before in any species. These relationships may be important for the interpretation of data concerning the coupling of the circular layers and a possible influence of both submucosal nerves and axons from Auerbach’s plexus on the circular muscle. The functional and immunochemical significante of the close association between ICC and the so-called synapselike specializations has yet to be clarified. Further studies are also necessary to test whether ICC located at the leve1 of Auerbach’s plexus are coupled to ICC-DMP and whether ICC-DMP may be involved in the coupling of the parallel lamellae of circular muscle, which are very wel1 developed in humans. A corresponding plexus of ICC located at the submucosal border of the external colonic musculature has been described in several speciesz4-” Electrophysiological evidente from canine colon suggests that these cells are colonic pacemaker cells.28*2gThey are innervated by vasoactive intestinal polypeptidecontaining nerves and may mediate inhibitory input to the colonic circular muscle.30-32 Similar studies on the smal1 intestine are lacking. Vasoactive intestinal polypeptide is preferentially located in the DMP in canine ileum,” and, in analogy with the findings in canine colon, it is possible that DMP exerts inhibitory input to the circular muscle through innervation of ICC. Electrophysiological studies on this region of the smal1 boweP4 have suggested that nonneural cells located between the main muscle layers and between the outer and inner subdivisions of the circular layer generate slow waves in several species, including humans. Although studies on human smal1 intestine are preliminary,14 it is therefore also possible that ICC-DMP may act in concert with ICC located at the leve1 of Auerbach’s plexus as smal1 intestinal pacemaker ce11s.gJo,33*34 It has been speculated that the region between the two circular layers would be an optimal position for external muscular mechanoreceptors.5s6**J6 The ultrastructure of mechanosensitive neurons and target cells in the circular muscle is unknown, but terminals of intrinsic afferent neurons may contact ICC both at the levels of Auerbach’s plexus and in the DMP. Vagal tension receptors are located in the ex-
67
ternal muscle layers,35 but the morphology and the distribution of these nerves and their target cells is also unknown. The morphology and the distribution of ICC fits wel1 with physiological concepts of tension receptor localization and shape.8,‘6*3” In conclusion, the present study has provided an ultrastructural description of a separate myoid cel1 type located in the innermost zone of the external muscle of the human smal1 intestine. These cells correspond to ICC-DMP of other mammals, and although the organization appears more diffuse, the cytology and organization of ICC-DMP is compatible with regulatory functions also in human smal1 intestine. The present study provides a basis for the interpretation of future physiological and pathological studies of this area of the human smal1 intestine. References 1. Cajal SR. Histologie
du système nerveux de 1’Homme et des Vertébrés. Paris: Maloine, 1911. Über die Struktur des Zir2. Li PL. Neue Beobachtungen kulärmuskels in Dünndarm bei Wirbeltieren. Eine vergleichende Studie. Z Anat Entw Gesch 1937;107:212-222. 3. Li PL. The intramural nervous system of the smal1 intestine with special reference to the innervation of the inner subdivision of its circular muscle. J Anat 1940;74:34a-359. 4. Gabella G. Innervation of the intestinal muscular coat. J Neurocytol 1972;1:341-362. 5. Gabella G. Special muscle cells and their innervation in the smal1 intestine. Cel1 Tissue Res 1974;153:63-77. 6. Duchon G, Henderson R, Daniel EE. Circular muscle layers in the smal1 intestine. In: Daniel EE, ed. Proceedings of the 4th International Symposium of Gastrointestinal Motility, Banff, Alberta, Canada: Mitchell, 1974:635-646. 7. Rumessen JJ, Thuneberg L. Plexus muscularis profundus and associated interstitial cells. 1. Light microscopical studies of mouse smal1 intestine. Anat Rec 1982;203:115-127. 8. Rumessen JJ, Thuneberg L, Mikkelsen HB. Plexus muscularis profundus and associated interstitial cells. 11.Ultrastructural studies of mouse smal1 intestine. Anat Rec 1982;203:129-146. 9. Thuneberg L. Interstitial cells of Cajal: intestinal pacemaker cells? Adv Anat Embryo1 Cel1 Bio1 1982;71:1-130. 10. Thuneberg L. Interstitial cells of Cajal. In: Wood JD, ed. Handbook of physiology, the gastrointestinal system. Volume 1. Bethesda, MD: American Physiological Society, 1989:349-386. ll. Berezin 1, Sheppard S, Daniel EE, Yanaihara N. Ultrastructural immunocytochemical distribution of VIP-like immunoreactivity in dog ileum. Regul Pept 1985;11:287-298. 12. Wilson AJ, Llewellyn-Smith IJ, Furness JB, Costa M. The source of nerve fibers forming the deep muscular and circular muscle plexuses in the smal1 intestine of the guinea pig. Cel1 Tissue Res 1987;247:497-504. 13. Llewellyn-Smith IJ, Furness JB, Gibbins IL, Costa M. Quantitative ultrastructural analysis of enkephalin-substance P-, and VIP-immunoreactive nerve fibers in the circular muscle of the guinea pig smal1 intestine. J Comp Neurol 1988; 272:139-148. 14. Hara Y, Kubota M, Szurszewski JH. Electrophysiology of smooth muscle of the smal1 intestine of some mammals. J Physiol 1986;372:501-520. 15. Yamamoto M. Electron microscopic studies on the innervation of the smooth muscle and the interstitial cel1 of Calal in
68
RUMESSEN ET AL.
the smal1 intestine of the mouse and bat. Arch Histol Jpn 1977;40:171-201. 16. Daniel EE. Nerves and motor activity of the gut. In: Brooks FP, Evers PW, eds. Nerves and the gut, Thorofare, NJ: Slack,
GASTROENTEROLOGY Vol. 102. No. 1
28. Barajos-Lopez
29.
1977:154-196. 17. Faussone-Pellegrini
18.
19.
20.
21.
22.
23.
24.
25.
26.
27.
MS, Cortesini C. Some ultrastructural features of the muscular coat of the human smal1 intestine. Acts Anat 1983;115:47-68. Rumessen JJ, Thuneberg L. Interstitial cells of Cajal in human smal1 intestine. Ultrastructural identification and organization between the main smooth muscle layers. Gastroenterology 1991;100:1417-1431. Mikkelsen HB, Thuneberg L, Rumessen JJ, Thorball N. Macrophage-like cells in the muscularis externa of the mouse smal1 intestine. Anat Rec 1985;213:77-86. Faussone-Pellegrini MS. Morphogenesis of the special circular muscle layer and of the interstitial cells of Cajal related to the plexus muscularis profundus of mouse intestinal muscle coat. An EM study. Anat Embryo1 1984;169:151-158. Kobayashi S, Suzuki M, Nishisaka T. Severed nerve stumps around a laser-irradiated lotus in the deep muscular plexus of the guinea-pig smal1 intestine. Arch Histol Cytol 1989; 52(Suppl):147-159. Kobayashi S, Furness JB, Smith T, Pompolo S. Histological identification of the interstitial cells of Cajal in the guinea-pig smal1 intestine. Arch Histol Jpn 1989;52:267-286. Tranum-Jensen J. The fine structure of the sinus node: a survey. In: Bonk FIM, ed. The sinus node. Structure, function and clinical relevante. Dordrecht, The Netherlands: Nijhoff, 1978:149-165. Stach W. Der Plexus Entericus Extremus des Dickdarmes und Seine Beziehungen zu den Interstitiellen Zellen (Cajal). Z Mikrosk Anat Forsch 1972;85:245-272. Faussone-Pellegrini MS. Ultrastructural peculiarities of the inner portion of the circular layer of the colon. Research in the mouse. Acts Anat 1985;122:187-192. Christensen J, Riek GA. Intrinsic nerves in the mammalian colon: confirmation of a plexus at the circular muscle-submucosal interface. J Auton Nerv Syst 1987;21:223-231. Berezin 1, Huizinga JD, Daniel EE. Interstitial cells of Cajal in the canine colon: a special communication network at the inner border of the circular muscle. J Comp Neurol 1988;273:42-51.
30.
31.
32.
33.
34.
35.
36.
C, Berezin 1, Daniel EE, Huizinga JD. Pacemaker activity recorded in interstitial cells of the gastrointestina1 tract. Am J Physiol 1989;257:C830-C835. Langton P, Ward SM, Carl A, Norell MA, Sanders KM. Spontaneous electrical activity of interstitial cells Cajal isolated from canine proximal colon. Proc Nat1 Acad Sci USA 1989;86:7280-7284, Berezin 1, Huizinga JD, Farraway L, Daniel EE. Innervation of interstial cells of Cajal by vasoactive intestinal polypeptide containing nerves in canine colon. Can J Physiol Pharmacol 1990;68:922-932. Smith TK, Reed JB, Sanders KM. Electrical pacemakers of canine proximal colon are functionally innervated by inhibitory motor neurons. Am J Physiol 1989;252:C466-C477. Huizinga JD, Berezin 1, Daniel EE, Chow E. Inhibitory innervation of colonic smooth muscle cells and interstitial cells of Cajal. Can J Physiol Pharmacol 1990;68:447-454. 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. Thuneberg L, Johansen V, Rumessen JJ, Andersen BG. Interstitial cells of Cajal: selective uptake of methylene blue inhibits slow wave activity. In: Roman C, ed. Gastrointestinal motility. Lancaster, England: MTP, 1984,495-502. Iggo A. Gastro-intestinal tension receptors with unmyelinated afferent fibres in the vagus of the cat. Q J Exp Physiol 1957;42:130-143. Leek BF. Abdominal visceral receptors. In: Neil E, ed. Handbook of sensory physiology. Volume 3. New York: Springer, 1972:113-129.
Received November 8, 1990. Accepted April 23,199l. Address requests for reprints to: Dr. Jiiri Johannes Rumessen, Department of Anatomy C, University of Copenhagen, Panum Instituttet, Blegdamsvej 3, DK-2200 Copenhagen, Denmark. Supported by The Novo Foundation and Holger Hjortenbergs Fond. The authors thank S. Peters, K. Stub-Christensen, and V. Heidemann for technical assistance. The helpful assistance of the staff of Department of Surgery D, Gentofte Hospital, and of Department of Surgery C, Rigshospitalet, is gratefully acknowledged.
