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The deep muscular plexus of the pig duodenum: A histochemical and ultrastructural study with special reference to the interstitial cells
Journal of the Autonomic Nervous System 70 Ž1998. 145–156
The deep muscular plexus of the pig duodenum: A histochemical and ultrastructural study with special reference to the interstitial cells M. Henry ) , C. Porcher, Y. Jule´ Departement de Physiologie et Neurophysiologie, CNRS-ESA 6034, Faculte´ des Sciences de Saint-Jerome, ´ ´ ˆ 13397 Marseille Cedex 20, France Received 27 August 1997; revised 16 January 1998; accepted 9 February 1998
Abstract The aim of the present study was to describe the deep muscular plexus of the pig duodenum and to characterize its cellular components. Numerous nerve varicosities have been detected in the deep muscular plexus using anti-synaptophysin antibodies. Nerve fibres were also detected here in the outer circular muscle layer, whereas no nerve fibres were observed in the inner circular muscle layer. In the deep muscular plexus, nerve fibres projected to interstitial cells which were characterized at the ultrastructural level. The interstitial cells were of two kinds: the interstitial fibroblastic-like cells ŽFLC. and the interstitial dense cells ŽIDC., both of which were interposed between nerve fibres and smooth muscle cells. The FLC were characterized by their elongated bipolar shape, the lack of basal lamina, a well-developed endoplasmic reticulum, a Golgi apparatus, and intermediate filaments. They were closely apposed to axon terminals containing small clear synaptic vesicles andror dense-cored vesicles. They were frequently connected to each other and to smooth muscle cells of the inner and outer circular layer by desmosomes and more rarely by gap junctions. The IDC are myoid-like cells. They had a stellate appearance and were characterized by a dense cell body, numerous caveolae, and a discontinuous basal lamina. The IDC were always closely apposed to nerve fibres and were connected to smooth muscle cells by desmosomes and small gap junctions. The present results show the unique pattern of cellular organization of the deep muscular plexus of the pig small intestine. They suggest that the interstitial cells in the deep muscular plexus are involved in the integration and transmission of nervous inputs from myenteric neurons to the inner and outer circular muscle layers. The clear-cut distinction observed here between the two types of interstitial cells Žfibroblasticand myoid-like. suggests that the interstitial cells of each type may also be involved in some other specific activity, which still remains to be determined. q 1998 Elsevier Science B.V. All rights reserved. Keywords: Cells of Cajal; Smooth muscle; Small intestine; Immunofluorescence; Electron microscopy; Pig
1. Introduction The deep muscular plexus ŽDMP., or plexus muscularis profundus ŽCajal, 1911., is an aganglionic nervous plexus which is located between the thick outer and thin inner layer of circular muscle cells of the small intestine ŽCajal, 1893; Li, 1940.. Physiological studies have emphasized the potential importance of the DMP in the control of the small intestinal motility ŽHara et al., 1986.. There exists growing evidence that this control is exerted through specialized populations of cells, referred to as interstitial cells ŽICs., which are involved directly in the generation of electrical rhythmicity and transmission of neural inputs within the circular muscle layer ŽTorihashi et al., 1995.. ) Corresponding author. Tel.: q33 04 91288759; fax: q33 04 91288759; e-mail: [email protected]
0165-1838r98r$19.00 q 1998 Elsevier Science B.V. All rights reserved. PII S 0 1 6 5 - 1 8 3 8 Ž 9 8 . 0 0 0 3 9 - 3
Ultrastructural studies on mice ŽFaussone-Pellegrini, 1984; Rumessen et al., 1982; Yamamoto, 1977., bats ŽYamamoto, 1977., rats ŽFaussone-Pellegrini, 1982, 1983; Komuro and Seki, 1995., guinea-pigs ŽGabella, 1972; Wilson et al., 1987; Zhou and Komuro, 1992a,b., cats ŽSilva, 1971., dogs ŽDuchon et al., 1974; Torihashi et al., 1993. and humans ŽFaussone-Pellegrini and Cortesini, 1983; Rumessen and Thuneberg, 1991., have shown that the ICs of the DMP have some characteristics in common: they are always located in the collagen, interposed between the nerve fibres and the smooth muscle cells; they establish close contacts with nerve fibres and are connected to smooth muscle cells by nexuses. The ICs of the DMP also have some particular characteristics however since they constitute a distinct set of heterogeneous cells differing in both their shape Žone to four different categories. and their number depending on the species studied.
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In the pig, investigations on the enteric nervous system have dealt mainly with the ganglionated nervous plexus ŽGunn, 1968; Scheuermann et al., 1989, 1987a,b; Scheuermann and Timmermans, 1993., whereas little information is available about the DMP ŽScheuermann et al., 1989.. The aim of the present study was first to further analyse the organization of the DMP with a view to understanding more clearly how the nervous control of motility operates in the pig small intestine. Secondly, it was proposed to examine the question as to whether the findings of the present study can be extrapolated to humans, since the human digestive system resembles that of the pig much more than that of the small laboratory animals mainly investigated so far. The nerve fibres present in the DMP were detected using fluorescence microscopy techniques, by means of synaptophysin antibodies known to label the small synaptic vesicles present in almost all nerve varicosities ŽNavone et al., 1986.. On the other hand, electron microscopic procedures were used in order to obtain an ultrastructural picture of the interstitial cells present in the DMP and to study their relationships with nerve fibres and the smooth muscle cells.
for 1 h at room temperature. Lastly, the sections were rinsed in PBS and coverslipped with an anti-fading mounting medium ŽDako.. They were examined 24 h later with an epifluorescence microscope equipped with appropriate filter blocks for examining Texas-red Žexcitation filters BP 530–560 nm, barriers LP 580 nm.. The specificity of the immunolabelling was checked by omitting the primary antiserum, which led to the complete absence of immunolabelling. The distribution of the synaptophysin-like immunoreactivity was observed in 10 sections randomly selected from the duodenum of each of 8 animals; the density of the synaptophysin-like immunoreactivity was indicated by a vertical picture of the grey level histograms giving the amounts of immunoreactive material present. This picture was obtained by digitalizing a representative histological section using a special computer software program ŽBiosys Laboratory, Marseille.. The intensity of each pixel, which ranged between 0 and 255 grey levels, was proportional to the number of fluorescent photons emitted by the corresponding point in the histological section. 2.3. Electron microscopy
2. Materials and methods 2.1. Animal preparation Experiments were carried out on 8 young Ž4 to 6 weeks old. pigs ŽPietrainrHampshire. of both sexes Ž5 males and 3 females. ranging from 6 to 8 kg in weight. Anaesthesia was induced with pentobarbital Ž10 mg kgy1 i.p.. and the animals were sacrificed by exsanguination. After a medioventral laparotomy, pieces of the duodenum were removed, immediately rinsed in Tyrode’s solution, and fixed for either fluorescence or electron microscopy. 2.2. Immunohistochemistry The intestinal segments were fixed for 24 h at 48C by immersing them in 4% paraformaldehyde in 0.1 M phosphate buffer ŽpH 7.4.. The tissues were then immersed for 24 h in a solution containing 20% sucrose in 0.1 M phosphate buffer ŽpH 7.4. and 30% sucrose for 48 h at 48C. They were finally immersed in a solution containing 30% sucrose, 5% glycerol and 0.1% azide for 48 h at 48C. The specimens were frozen in CO 2 and cut on a cryostat into sections with a thickness of 20 m m. The sections were first treated in 0.15 M phosphate buffer saline ŽPBS. containing 3% normal goat serum and 0.2% Triton X100 for 1 h 30 min at room temperature. They were then incubated with a monoclonal mouse anti-synaptophysin serum ŽSera-Lab. diluted 1:15 in PBS containing 1% normal goat serum at 48C for 24 h. After being rinsed in PBS, the sections were incubated with Texas-red conjugated goat anti-mouse IgG ŽJackson. at a dilution of 1:100
Tissues were immersed in a solution of 2% glutaraldehyde in 0.1 M Sorensen’s phosphate buffer ŽpH 7.4. for 1 ¨ h at 48C. After being rinsed in phosphate buffer several times for 30 min, the specimens were postfixed in 2% osmium tetroxide in 0.1 M Sorensen’s phosphate buffer ¨ ŽpH 7.4. for 30 min at 48C. They were then dehydrated in a graded series of ethyl alcohols, immersed in propylene oxide for 30 min at room temperature and finally embedded in Epon. The specimens were cut into semithin sections Ž0.3–0.5 m m. which were stained with Azure blue, and ultrathin sections, which were contrasted with uranyl acetate and lead citrate. The semithin sections were examined in a photonic microscope ŽAristoplan, Leitz. and the ultrathin sections in an electron microscope ŽPhilips EM 400T..
3. Results 3.1. Light microscopy The DMP of the pig duodenum was located between the inner and outer circular muscle layer ŽFig. 1A, B.. The inner circular muscle layer was a very thin layer containing 2–3 strata of smooth muscle cells. These cells were smaller and denser than those of the outer circular layer, which was about ten times thicker. The DMP consisted of a cluster of non-neuronal cells and nerve fibres, connected together by networks consisting mainly of nerve fibres and a few rare non-neuronal cells. When cutting sections, one therefore crossed at random through either the tracts ŽFig. 1A. or the cell clusters ŽFig. 1B..
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Fig. 1. Photomicrographs of semithin sections of the pig duodenum. The deep muscular plexus was composed of clusters of non-neuronal cells ŽB, arrows. linked together by thin tracts ŽA, arrows.. This plexus was located between the outer ŽOCM. and inner ŽICM. circular muscle layer. The inner circular muscle layer was separated from the external submucous plexus ŽESP. by loose connective tissue. The outer circular muscle layer was separated from the longitudinal muscle layer ŽLM. by the myenteric nervous plexus ŽMP..
3.2. Immunohistochemistry
3.3. Electron microscopy
In all the sections examined, the synaptophysin-like immunoreactivity was located in the ganglionated plexus, myenteric and external submucous plexus, the varicose nerve fibres present in the outer circular muscle layer and in the DMP; no immunoreactivity was revealed in the inner circular muscle layer ŽFig. 2A.. In order to illustrate the density of the immunoreactive material shown in Fig. 2A, the data in this figure were digitized in order to obtain a vertical picture of the grey level histograms ŽFig. 2B.. The pattern of distribution of the grey level histograms indicated that the density of the synaptophysin-like immunoreactivity was particularly high in the ganglionated plexus and the DMP.
Our ultrastructural study on the pig DMP showed the presence of various nerve fibres, Schwann cells, macrophages and interstitial cells. 3.3.1. Schwann cells The Schwann cells were sometimes surrounded by non-myelinated nerve fibres ŽFig. 3A.. They had a central nucleus containing both marginal and perinucleolar heterochromatin ŽFig. 3A.. Their cytoplasm formed a thin perinuclear rim with long extensions. The perinuclear cytoplasm contained numerous ribosomes, a few mitochondria, a few endoplasmic rough reticulum, one centriole, a poorly
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Fig. 2. Synaptophysin-like immunoreactivity in the pig duodenum. ŽA. A conventional fluorescence photomicrograph of the synaptophysin immunoreactivity present in varicose nerve fibres distributed throughout the duodenal wall is shown. It can be noted that the density of the immunoreactivity was particularly high in the myenteric Žmp. and deep muscular plexus Žarrows.. ŽB. A vertical picture of the grey level histograms of the synaptophysin immunoreactivity given in ŽA. is shown. This picture was obtained by digitalizing of the photomicrograph in ŽA.; the grey level histograms are indicated by means of artificial colors. LM: longitudinal muscle; OCM: outer circular muscle; ICM: inner circular muscle; SM: submucosae; mp: myenteric plexus; esp: external submucous plexus.
developed Golgi apparatus and some rare lysosomes. The cytoplasmic extensions enveloped numerous nerve fibres. Microtubules and gliofilaments were present throughout the cytoplasm. The Schwann cells were always surrounded by a continuous thick basal lamina.
3.3.2. NerÕe fibres The nerve fibres contained vesicles of several kinds: either small clear synaptic vesicles ŽFig. 4B., or clusters of small dense vesicles ŽFig. 3C., or more frequently heterogeneous populations including dense-cored vesicles, small
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Fig. 3. Electron photomicrograph of Schwann cells ŽSC. of the deep muscular plexus of the pig duodenum ŽA.; these cells were located inside the deep muscular plexus located between the inner ŽICM. and the outer ŽOCM. circular muscle layers. The Schwann cells were delimited by a continuous basal lamina ŽBL. closely surrounding the nerve fibres ŽNF.. Electron photomicrographs of nerve fibres ŽNF. of the deep muscular plexus of the pig duodenum ŽB and C.. ŽB. A nerve fibre in contact with a fibroblastic-like cell ŽFLC. is shown; note the presence in the nerve fibre of small clear vesicles Žsmall arrows., large dense-cored vesicles Žlarge arrow., small areolated vesicles Žhead arrows. and large granular vesicles Žstar.. ŽC. One of the nerve fibres, facing the inner circular muscle layer ŽICM. contained similar numerous dense-cored vesicles, while in the other fibre, different types of vesicles were observed. Scale bar: 0.4 m m.
clear vesicles, small areolated vesicles and large granular vesicles ŽFig. 3B.. All these nerve fibres were arranged parallel to the adjacent smooth muscle cells and were often closely apposed to the two types of ICs described below.
3.3.3. Interstitial cells The ICs present in the DMP were of two kinds, namely interstitial fibroblastic-like cells ŽFLC. ŽFig. 4A. and interstitial dense cells ŽIDC. ŽFig. 4B..
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Fig. 4. Electron photomicrographs of a fibroblastic-like cell and interstitial dense cell of the deep muscular plexus of the pig duodenum. ŽA. Note that the fibroblastic-like cell ŽFLC. was apposed to a nerve fibre ŽNF., both of which were delimited by the inner ŽICM. and outer ŽOCM. circular muscle layers; ŽB. An interstitial dense cell ŽIDC. characterized by a dense heterochromatin ŽH. nucleus ŽN. with a voluminous nucleolus ŽNu., a cytoplasm containing numerous ribosomes and caveolae Žarrows. is shown; note the presence of a nerve fibre ŽNF. containing numerous small clear vesicles, closely apposed to the interstitial cell. Scale bar: 0.4 m m.
The FLC always ran parallel to the smooth muscle cells and were electron-clear and bipolar ŽFig. 4A, Fig. 5A.. They had a central ovoid nucleus with an irregular contour, which was rimmed by a fairly regular heterochromatin border and contained a voluminous nucleolus ŽFig. 4A..
The FLC were characterized by an abundant endoplasmic rough reticulum containing a medium electron-dense fibrillar component, and numerous mitochondria ŽFig. 5A.. Islets consisting of smooth endoplasmic reticulum tubules of dense material were also present ŽFig. 5B.. In some
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Fig. 5. Electron photomicrographs of fibroblastic-like cells ŽFLC. of the deep muscular plexus of the pig duodenum, interposed between the inner ŽICM. and outer ŽOCM. circular muscle layer. In the FLC, note the presence of abundant endoplasmic rough reticulum ŽE. surrounding mitochondria ŽM., tubules of smooth endoplasmic reticulum ŽSER., lysosomes ŽL. and ribosomes ŽR.. Scale bar: 0.4 m m.
places, voluminous secondary lysosomes had accumulated, as well as numerous free ribosomes ŽFig. 5C.. In the vicinity of the nucleus, the Golgi apparatus could be observed ŽFig. 6A.. The latter was characterized by the presence of a few sacculae and a very large number of vesicles, some of which were coated vesicles. A cilium
was sometimes observed in the vicinity of the Golgi apparatus ŽFig. 6A.. The FLC were found to be the sites of intensive pinocytic activity ŽFig. 6A.. The FLC were frequently connected together by interdigitations ŽFig. 6B.. They did not contain any basal lamina and were much more numerous than the IDC. The FLC were interposed
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Fig. 6. Electron photomicrographs of fibroblastic-like cells ŽFLC. of the deep muscular plexus of the pig duodenum. The FLC were characterized by a Golgi apparatus ŽG. close to the nucleus, a pinocytic ŽA, arrow. activity and a cilium ŽC.. They were connected together by interdigitations ŽB, arrow., desmosomes ŽB, head arrows, and E, arrow. and gap junction ŽC, arrow.; they were in close position with nerve fibers ŽNF.Ž6D.. Scale bar: 0.4 m m.
between nerve endings and smooth muscle cells belonging to either the inner or outer circular muscle layer. The nerve endings were linked to the FLC by membrane apposition only ŽFig. 6D., whereas the FLC were connected to each other and to smooth muscle cells by desmosomes ŽFig. 6B, E. and more rarely by gap junctions ŽFig. 6C.; among 50
sections from 50 different FLC, only two gap junctions were identified, while 30 desmomes were observed. The IDC were either stellate ŽFig. 7A. or bipolar ŽFig. 7B, C. and ran parallel to the smooth muscle cells ŽFig. 4B, Fig. 7B, C.. They had a multi-lobed nucleus which was characterized by large amounts of marginal and peri
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Fig. 7. Electron photomicrographs of interstitial cells of the deep muscular plexus of the pig duodenum. The interstitial dense cells ŽIDC. were located between the inner ŽICM. and outer ŽOCM. circular muscle layer. They were often close to the fibroblastic-like cells ŽFLC.. Note that the IDC were interposed between a nerve fibre ŽNF. and a smooth muscle cell ŽMC.. The IDC were characterized by a discontinuous basal lamina ŽFig. 7, arrows., large mitochondria ŽM. ŽA and B., a Golgi apparatus ŽG. ŽA., an endoplasmic rough reticulum ŽE. which was often dilated ŽA and C., numerous ribosomes ŽR. ŽC., polysomes ŽP. ŽB., and dense bodies ŽC, arrows.. Note that IDC were connected to the adjacent smooth muscle cell by small gap junctions ŽD, arrows.. Scale bar: 0.4 m m.
nucleolar heterochromatin ŽFig. 4B, Fig. 7B, C. and a voluminous nucleolus ŽFig. 4B.. The cytoplasm was found to contain a few large mitochondria, numerous ribosomes and polysomes ŽFig. 4B, Fig. 7B, C., endoplasmic rough
reticulum with some dilated cisternae ŽFig. 7A, C., and a Golgi apparatus ŽFig. 7A.. The IDC were furthermore characterized by the presence of dense bodies ŽFig. 7C., numerous caveolae ŽFig. 4B, Fig. 7B, D., a discontinuous
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basal lamina ŽFig. 7A., and the occurrence of pinocytic activity ŽFig. 7B.. The IDC were interposed between nerve fibres to which they were closely apposed ŽFig. 4B, Fig. 7A. and smooth muscle cells to which they were connected by gap junctions ŽFig. 7D.. The presence of ICs was furthermore observed in the interlamellar septa, in the aganglionic tracts of the outer circular muscle layer, and in the plexus muscularis superficialis. The FLC predominated in the DMP and the external circular muscle layer, and the IDC in the septa and the plexus muscularis superficialis. No nerve fibres or ICs were detected in the inner circular muscle layer.
4. Discussion To our knowledge, this is the first detailed ultrastructural study to be carried out so far on the deep muscular plexus of the pig duodenum. The main histological features of the pig duodenal DMP observed in the present study were broadly similar to those of the small intestine of the other species studied so far, namely the guinea-pig ŽGabella, 1972; Wilson et al., 1987; Zhou and Komuro, 1992a,b., bat ŽYamamoto, 1977., rat ŽKomuro and Seki, 1995., mouse ŽRumessen et al., 1982; Yamamoto, 1977., dog ŽTorihashi et al., 1993., and Man ŽFaussone-Pellegrini and Cortesini, 1983.. In addition, these histological features of small intestine DMP are also similar to those of the plexus entericus extremus described in the colon of the cat ŽConklin and Du, 1990., dog ŽLiu et al., 1994; Serio et al., 1991. and Man ŽFaussone-Pellegrini, 1992; Faussone-Pellegrini et al., 1990.. The main cellular components of the pig duodenal DMP were found to belong to the same broad categories of cells as those described in other species, since they consist of macrophages, Schwann cells, nerve fibres and ICs. In the present study, the analysis of synaptophysin-like immunoreactivity clearly showed that a large number of varicose nerve fibres present in the DMP contained small clear vesicles. The existence of these vesicles was further confirmed at the ultrastructural level. Our ultrastructural results show, however, that other types of vesicles are often co-localized with these small clear vesicles. In the DMP of the guinea-pig, the small clear vesicles contain either VIP or SP, sometimes along with large granular enkephalin-containing vesicles; VIP and SP may be involved in the transmission of inhibitory and excitatory inputs, respectively to smooth muscle, whereas enkephalins may modulate both excitatory and inhibitory nerve fiber transmission ŽLlewellyn-Smith et al., 1988.. Pig myenteric neurons were also found to contain VIP, SP and enkephalins as well as galanin, CGRP, somatostatin, NPY and serotonin ŽTimmermans et al., 1991.. The presence of enkephalin-like immunoreactive fibres has been previously demonstrated in the pig DMP ŽHenry et al., 1996.. It has been suggested that the DMP nerves in the pig may
correspond to the Dogiel’s type II neurons located in the myenteric plexus ŽScheuermann et al., 1989.. It is therefore possible that transmitters present in nerve fibres of the pig DMP may have similar subcellular localizations and functions to those described in the guinea-pig ŽLlewellynSmith et al., 1988.. The lack of any anatomical connecti vity between the DMP and the external submucosal plexus observed in this study strongly suggests that all the DMP nerve fibres may originate from the myenteric plexus as previously found to occur in the guinea-pig ŽWilson et al., 1987.. It is worth noting that a number of nerve strands run from the myenteric plexus to the external submucous plexus ŽScheuermann et al., 1987b; unpublished data.. This pattern of organization of the intramural pathways strongly suggests the existence of peripheral reflex loops between the external submucous plexus, the myenteric plexus and the DMP. The results of the present study show that in the pig DMP the nerve fibres run between ICs of two main types: those of the FLC type, which predominated, and those of the IDC type. This overall picture suggests that FLC may therefore be ‘interstitial cells’, which were defined in the review by Komuro Ž1990. as originating from fibroblasts but being differentiated depending on the neighbouring structures, nerves and muscles. The FLC present in the pig DMP were similar to those of the rat DMP, as well as to the ZIO positive ICs observed in the guinea-pig DMP ŽZhou and Komuro, 1992a,b, 1995.. The present results indicate however that in the pig DMP, the FLC never contain any of the basal lamina or glycogen particles present in the other group of FLC described in the guineapig DMP ŽZhou and Komuro, 1992a,b, 1995.. The existence of a cilium has also been described in mouse FLC, but its functional role still remains to be determined ŽRumessen et al., 1982.. The main characteristics of the IDC observed here in the pig DMP indicate that they are of the myoid-like type and tend to resemble the ICs described in the lovebird ŽImaizumi and Hama, 1969., dog ŽDuchon et al., 1974; Torihashi et al., 1993., bat ŽYamamoto, 1977., mouse ŽRumessen et al., 1982; Yamamoto, 1977., rat ŽKomuro and Seki, 1995., guinea-pig ŽZhou and Komuro, 1992b., and Man ŽFaussone-Pellegrini, 1983. where they are the only type of ICs present. The pig ICs resemble those described in Man, however, in that they contain only very few small gap junctions, whereas the ICs of the other species listed above contain a large number of long gap junctions. The IDC might correspond to a non-differen tiated or immature type of smooth muscle cell ŽYamamoto, 1977.. Both types of ICs detected here in the pig DMP were closely apposed to nerve fibres. Gabella Ž1972. has reported that the close appositions between ICs and nerves are actually connections which may mediate communications. In addition, we observed that the ICs in the pig DMP were connected by gap junctions not only to each other,
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but also to smooth muscle cells of both the inner and outer circular muscle layers. Although these gap junctions were small and sparse, the finding that connexin mRNA is present in the dog colon ŽLi et al., 1993. suggests that there may exist minute gap junctions which are not detectable at the electron microscopic level. Finally, the interstitial cells of Cajal are a population of cells in the gastrointestinal tract which play a role in the control of gut motility ŽHagger et al., 1997.; these cells are characterized by their appearance, which is either bipolar or stellate, their network arrangement, and by the fact that they are interposed between nerve fibres and smooth muscle cells. Both types of ICs cells described here in the pig DMP can therefore be said to be interstitial cells of Cajal. In this study, no innervation was detected in the inner circular muscle layer of the pig duodenum. In view of the particular smooth muscle cells of which it is composed, this layer probably has a specific function, as previously suggested ŽGabella, 1972.. In the pig, the inner circular muscle layer was contacted only by ICs of the DMP which were themselves connected to nerve fibres. Some authors ŽGabella, 1972. have concluded that the DMP may control only the inner circular muscle layer, while others ŽDuchon et al., 1974. favour the idea that it may control only the outer circular layer. The present results obtained on the pig suggest that the DMP may be involved in the control of both inner and outer circular muscle layers via the ICs. Finally, the data obtained on various species, including pigs, suggest that the role of the DMP in the control of the circular muscle layer probably differs from one species to another. The network of ICs in the pig DMP, which are interposed between nerves and smooth muscle cells, differ from the ICC–AP located in the myenteric plexus. In the pig Žunpublished data. as in the guinea-pig ŽZhou and Komuro, 1995. and Man ŽFaussone-Pellegrini et al., 1990., the ICC–AP constitute an independent cellular network, having only rare, if any, contacts with the nervous pathways. Moreover, in the small intestine of c-kit protooncogene mutant mice, it has been reported that the number of ICC–AP was considerably lower than in normal mice, while the smooth muscle electrical slow waves were completely absent ŽWard et al., 1994.. The mutation had no effect however on either the ICC–DMP or the excitatory and inhibitory nervous inputs to the smooth muscle cells. It has been suggested that in the small intestine of the mouse, the ICC–AP may be mainly involved in the pacemaker activity of smooth muscle cells ŽWard et al., 1994., while the ICC–DMP may mediate neural inputs ŽBurns et al., 1996.. The present finding that the ICs ŽFLC and IDC. of the pig DMP are interposed between nerve fibres and smooth muscle cells is consistent with the intercalation hypothesis first put forward by Cajal Ž1911., who suggested that these cells might play a fundamental role in neurotransmission. The ICs of the pig DMP may therefore be involved at least
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in the integration and conduction of excitatory and inhibitory nervous inputs from myenteric motoneurons to the inner and outer circular muscle layers. The clear-cut distinction found to exist in the present study between the two types of ICs Žfibroblastic- and myoid-like. present in the pig DMP indicates that the ICs of each type may also be involved in some other specific activity, which still remains to be determined. Acknowledgements The authors wish to express their sincere thanks to Christine Meyer and Jean Claude Stamegna for their excellent technical assistance. The authors would also like to thank Pierre Rage for his help with the computer image analysis. References Burns, A.J., Lomax, A.E.J., Torihashi, S., Sanders, K.M., Ward, S.M., 1996. Interstitial cells of Cajal mediate inhibitory transmission in the stomach. Proc. Natl. Acad. Sci. USA 93, 12008–12013. Cajal, S.R., 1893. Sur les ganglions et plexus nerveux de l’intestin. Comp. Rend. Sean. ´ Soc. Biol. 5, 217–223. Cajal, S.R., 1911. Histologie du systeme ` nerveux de l’Homme et des vertebres. ´ ´ Maloine ŽParis. 2, 891–942. Conklin, J.L., Du, C., 1990. Pathways of slow-wave propagation in proximal colon of cats. Am. J. Physiol. 258, G894–G903. Duchon, G.R., Henderson, E., Daniel, E., 1974. Circular muscle layers in the small intestine. In: Daniel, E. ŽEd.., Proceedings of the Fourth International Symposium on Gastrointestinal Motility, Mitchell Press, Vancouver, pp. 636–646. Faussone-Pellegrini, M.S., 1982. Ultrastuctura e topografia delle cellule interstiziali del Cajal nello strato circolare della tunica muscolare del ileo e del colon del ratto. Bol. Soc. Ital. Biol. Sper. 58, 1260–1265. Faussone-Pellegrini, M.S., 1983. Le caracteristiche ultrastructurali della tunica muscolare del ileo e del colon di ratto: A. Porzione interna della strato circolare. Arch. Ital. Anat. Embryol. 88, 25–40. Faussone-Pellegrini, M.S., 1984. 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 E.M. study. Anat. Embryol. ŽBerlin. 169, 151–158. Faussone-Pellegrini, M.S., 1992. Histogenesis, structure and relationships of interstitial cells of Cajal: from morphology to functional interpretation. Eur. J. Morphol. 30, 137–148. Faussone-Pellegrini, M.S., Cortesini, C., 1983. Some ultrastructural features of the muscular coat of human small intestine. Acta Anat. ŽBasel. 115, 47–68. Faussone-Pellegrini, M.S., Pantalone, D., Cortesini, C., 1990. Smooth muscle cells, interstitial cells of Cajal and myenteric plexus interrelationships in the human colon. Acta Anat. ŽBasel. 139, 31–44. Gabella, G., 1972. Innervation of the intestinal muscular coat. J. Neurocytol. 1, 341–362. Gunn, M., 1968. Histological and histochemical observations on the myenteric and submucous plexuses of mammals. J. Anat. 102, 223– 239. Hagger, R., Finlayson, C., Jeffrey, I., Kumar, D., 1997. Role of the interstitial cells of Cajal in the control of gut motility. Br. J. Surg. 84, 445–450. Hara, Y., Kubota, M., Szurszewski, J.H., 1986. Electrophysiology of smooth muscle of the small intestine of some mammals. J. Physiol. 372, 501–520.
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Henry, M., Porcher, C., Jule, ´ Y., 1996. Ultrastructural and immunohistochemical characteristics of the deep muscular plexus in the pig duodenum: an electron microscopy transmission and confocal microscopy study, Proceeding of the 11th European Congress of Electron Microscopy, GBM 79. Imaizumi, M., Hama, K., 1969. An electron microscopic study on the interstitial cells of the gizzard in the love-bird ŽUroloncha domestica.. Z. Zellforsch. 97, 351–357. Komuro, T., 1990. Re-evaluation of fibroblasts and fibroblast-like cells. Anat. Embryol. 182, 103–112. Komuro, T., Seki, K., 1995. Fine structural study of interstitial cells associated with the deep muscular plexus of the rat small intestine, with special reference to the intestinal pacemaker cells. Cell Tissue Res. 282, 129–134. Li, P.L., 1940. The intramural nervous system of the small intestine with special reference to the innervation of the inner subdivision of its circular muscle. J. Anat. 74, 348–359. Li, Z., Zhou, Z., Daniel, E.E., 1993. Expression of gap junction connexin 43 and connexin 43 mRNA in different regional tissues of intestine in dog. Am. J. Physiol. 265, G911–G916. Liu, L.W.C., Thuneberg, L., Huizinga, J.D., 1994. Selective lesioning of interstitial cells of Cajal by methylene blue and light leads to loss of slow waves. Am. J. Physiol. 266, G485–G496. Llewellyn-Smith, I.J., Furness, J.B., Gibbins, I.L., Costa, M., 1988. Quantitative ultrastructural analysis of enkephalin-, substance P-, and VIP-immunoreactive nerve fibers in the circular muscle of the guinea pig small intestine. J. Comp. Neurol. 272, 139–148. Navone, F., Jahn, R., Di Gioia, G., Stukenbrok, H., Greengard, P., De Camilli, P., 1986. Protein p38: An integral membrane protein specific for small vesicles of neurons and neuroendocrine cells. J. Cell Biol. 103, 2511–2527. Rumessen, J.J., Thuneberg, L., 1991. Interstitial cells of Cajal in human small intestine. Ultrastructural identification and organization between the main smooth muscle layers. Gastroenterology 100, 1417–1431. Rumessen, J.J., Thuneberg, L., Mikkelsen, H.B., 1982. Plexus muscularis profundus and associated interstitial cells: II. Ultrastructural studies of mouse small intestine. Anat. Rec. 203, 129–146. Scheuermann, D.W., Timmermans, J.P., 1993. Differing chemical content of the neuronal populations of submucosal ganglionic plexus of the enteric nervous system. Gastroenterology 104, 1570–1579. Scheuermann, D.W., Stach, W., Timmermans, J.P., 1987a. Topography, architecture and structure of the plexus submucosus internus ŽMeissner. of the porcine small intestine in scanning electron microscopy. Acta Anat. ŽBasel. 129, 96–104.
Scheuermann, D.W., Stach, W., Timmermans, J.P., 1987b. Topography, architecture and structure of the plexus submucosus externus ŽSchabadasch. of the porcine small intestine in scanning electron microscopy. Acta Anat. ŽBasel. 129, 105–115. Scheuermann, D.W., Stach, W., Adriaensen, D., De Groodt-Lasseel, M.H.A., 1989. Neuron-specific enolase and S-100 protein immunohistochemistry for defining the structure and topographical relationship of the different enteric nerve plexuses in the small intestine of the pig. Cell Tissue Res. 256, 65–75. Serio, R., Barajas-Lopez, C., Daniel, E.E., Berezin, I., Huizinga, J.D., 1991. Slow-wave activity in colon: role of network of submucosal interstitial cells of Cajal. Am. J. Physiol. 260, G636–G645. Silva, D.G., 1971. The fine structure and innervation of the inner circular muscle layer of the cat ileum. Anat. Rec. 169, 428–429. Timmermans, J.P., Adriaensen, D., Scheuermann, D.W., Stach, W., 1991. Morphological features of the enteric nervous system of an omnivorous animal, the domestic pig. J. Auton. Nerv. Syst. 33, 193–194. Torihashi, S., Kobayashi, S., Gerthoffer, W.T., Sanders, K.M., 1993. Interstitial cells in deep muscular plexus of canine small intestine may be specialized smooth muscle cells. Am. Physiol. Soc. 93, 1857–1930. Torihashi, S., Ward, S.M., Nishikawa, S., Nishi, K., Kobayashi, S., Sanders, K.M., 1995. C-kit-dependent development of interstitial cells and electrical activity in the murine gastrointestinal tract. Cell Tissue Res. 280, 97–111. Ward, S.M., Burns, A.J., Torihashi, S., Sanders, K.M., 1994. Mutation of the proto-oncogene c-kit blocks development of interstitial cells and electrical rhythmicity in murine intestine. J. Physiol. 480, 91–97. Wilson, A.J., Llewellyn-Smith, I.J., Furness, J.B., Costa, M., 1987. The source of the nerve fibres forming the deep muscular and circular muscle plexuses in the small intestine of the guinea-pig. Cell Tissue Res. 247, 497–504. Yamamoto, M., 1977. Electron microscopic studies on the smooth muscle and the interstitial cells of Cajal in the small intestine of the mouse and bat. Arch. Histol. Jpn. 40, 171–201. Zhou, D.S., Komuro, T., 1992a. Interstitial cells associated with the deep muscular plexus of the guinea-pig small intestine, with special reference to the interstitial cells of Cajal. Cell Tissue Res. 268, 205–216. Zhou, D.S., Komuro, T., 1992b. The cellular network of interstitial cells associated with the deep muscular plexus of the guinea pig small intestine. Anat. Embryol. ŽBerlin. 186, 519–527. Zhou, D.S., Komuro, T., 1995. Ultrastructure of the zinc iodide–osmic acid stained cells in guinea pig small intestine. J. Anat. 187, 481–485.
The deep muscular plexus of the pig duodenum: A histochemical and ultrastructural study with special reference to the interstitial cells M. Henry ) , C. Porcher, Y. Jule´ Departement de Physiologie et Neurophysiologie, CNRS-ESA 6034, Faculte´ des Sciences de Saint-Jerome, ´ ´ ˆ 13397 Marseille Cedex 20, France Received 27 August 1997; revised 16 January 1998; accepted 9 February 1998
Abstract The aim of the present study was to describe the deep muscular plexus of the pig duodenum and to characterize its cellular components. Numerous nerve varicosities have been detected in the deep muscular plexus using anti-synaptophysin antibodies. Nerve fibres were also detected here in the outer circular muscle layer, whereas no nerve fibres were observed in the inner circular muscle layer. In the deep muscular plexus, nerve fibres projected to interstitial cells which were characterized at the ultrastructural level. The interstitial cells were of two kinds: the interstitial fibroblastic-like cells ŽFLC. and the interstitial dense cells ŽIDC., both of which were interposed between nerve fibres and smooth muscle cells. The FLC were characterized by their elongated bipolar shape, the lack of basal lamina, a well-developed endoplasmic reticulum, a Golgi apparatus, and intermediate filaments. They were closely apposed to axon terminals containing small clear synaptic vesicles andror dense-cored vesicles. They were frequently connected to each other and to smooth muscle cells of the inner and outer circular layer by desmosomes and more rarely by gap junctions. The IDC are myoid-like cells. They had a stellate appearance and were characterized by a dense cell body, numerous caveolae, and a discontinuous basal lamina. The IDC were always closely apposed to nerve fibres and were connected to smooth muscle cells by desmosomes and small gap junctions. The present results show the unique pattern of cellular organization of the deep muscular plexus of the pig small intestine. They suggest that the interstitial cells in the deep muscular plexus are involved in the integration and transmission of nervous inputs from myenteric neurons to the inner and outer circular muscle layers. The clear-cut distinction observed here between the two types of interstitial cells Žfibroblasticand myoid-like. suggests that the interstitial cells of each type may also be involved in some other specific activity, which still remains to be determined. q 1998 Elsevier Science B.V. All rights reserved. Keywords: Cells of Cajal; Smooth muscle; Small intestine; Immunofluorescence; Electron microscopy; Pig
1. Introduction The deep muscular plexus ŽDMP., or plexus muscularis profundus ŽCajal, 1911., is an aganglionic nervous plexus which is located between the thick outer and thin inner layer of circular muscle cells of the small intestine ŽCajal, 1893; Li, 1940.. Physiological studies have emphasized the potential importance of the DMP in the control of the small intestinal motility ŽHara et al., 1986.. There exists growing evidence that this control is exerted through specialized populations of cells, referred to as interstitial cells ŽICs., which are involved directly in the generation of electrical rhythmicity and transmission of neural inputs within the circular muscle layer ŽTorihashi et al., 1995.. ) Corresponding author. Tel.: q33 04 91288759; fax: q33 04 91288759; e-mail: [email protected]
0165-1838r98r$19.00 q 1998 Elsevier Science B.V. All rights reserved. PII S 0 1 6 5 - 1 8 3 8 Ž 9 8 . 0 0 0 3 9 - 3
Ultrastructural studies on mice ŽFaussone-Pellegrini, 1984; Rumessen et al., 1982; Yamamoto, 1977., bats ŽYamamoto, 1977., rats ŽFaussone-Pellegrini, 1982, 1983; Komuro and Seki, 1995., guinea-pigs ŽGabella, 1972; Wilson et al., 1987; Zhou and Komuro, 1992a,b., cats ŽSilva, 1971., dogs ŽDuchon et al., 1974; Torihashi et al., 1993. and humans ŽFaussone-Pellegrini and Cortesini, 1983; Rumessen and Thuneberg, 1991., have shown that the ICs of the DMP have some characteristics in common: they are always located in the collagen, interposed between the nerve fibres and the smooth muscle cells; they establish close contacts with nerve fibres and are connected to smooth muscle cells by nexuses. The ICs of the DMP also have some particular characteristics however since they constitute a distinct set of heterogeneous cells differing in both their shape Žone to four different categories. and their number depending on the species studied.
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In the pig, investigations on the enteric nervous system have dealt mainly with the ganglionated nervous plexus ŽGunn, 1968; Scheuermann et al., 1989, 1987a,b; Scheuermann and Timmermans, 1993., whereas little information is available about the DMP ŽScheuermann et al., 1989.. The aim of the present study was first to further analyse the organization of the DMP with a view to understanding more clearly how the nervous control of motility operates in the pig small intestine. Secondly, it was proposed to examine the question as to whether the findings of the present study can be extrapolated to humans, since the human digestive system resembles that of the pig much more than that of the small laboratory animals mainly investigated so far. The nerve fibres present in the DMP were detected using fluorescence microscopy techniques, by means of synaptophysin antibodies known to label the small synaptic vesicles present in almost all nerve varicosities ŽNavone et al., 1986.. On the other hand, electron microscopic procedures were used in order to obtain an ultrastructural picture of the interstitial cells present in the DMP and to study their relationships with nerve fibres and the smooth muscle cells.
for 1 h at room temperature. Lastly, the sections were rinsed in PBS and coverslipped with an anti-fading mounting medium ŽDako.. They were examined 24 h later with an epifluorescence microscope equipped with appropriate filter blocks for examining Texas-red Žexcitation filters BP 530–560 nm, barriers LP 580 nm.. The specificity of the immunolabelling was checked by omitting the primary antiserum, which led to the complete absence of immunolabelling. The distribution of the synaptophysin-like immunoreactivity was observed in 10 sections randomly selected from the duodenum of each of 8 animals; the density of the synaptophysin-like immunoreactivity was indicated by a vertical picture of the grey level histograms giving the amounts of immunoreactive material present. This picture was obtained by digitalizing a representative histological section using a special computer software program ŽBiosys Laboratory, Marseille.. The intensity of each pixel, which ranged between 0 and 255 grey levels, was proportional to the number of fluorescent photons emitted by the corresponding point in the histological section. 2.3. Electron microscopy
2. Materials and methods 2.1. Animal preparation Experiments were carried out on 8 young Ž4 to 6 weeks old. pigs ŽPietrainrHampshire. of both sexes Ž5 males and 3 females. ranging from 6 to 8 kg in weight. Anaesthesia was induced with pentobarbital Ž10 mg kgy1 i.p.. and the animals were sacrificed by exsanguination. After a medioventral laparotomy, pieces of the duodenum were removed, immediately rinsed in Tyrode’s solution, and fixed for either fluorescence or electron microscopy. 2.2. Immunohistochemistry The intestinal segments were fixed for 24 h at 48C by immersing them in 4% paraformaldehyde in 0.1 M phosphate buffer ŽpH 7.4.. The tissues were then immersed for 24 h in a solution containing 20% sucrose in 0.1 M phosphate buffer ŽpH 7.4. and 30% sucrose for 48 h at 48C. They were finally immersed in a solution containing 30% sucrose, 5% glycerol and 0.1% azide for 48 h at 48C. The specimens were frozen in CO 2 and cut on a cryostat into sections with a thickness of 20 m m. The sections were first treated in 0.15 M phosphate buffer saline ŽPBS. containing 3% normal goat serum and 0.2% Triton X100 for 1 h 30 min at room temperature. They were then incubated with a monoclonal mouse anti-synaptophysin serum ŽSera-Lab. diluted 1:15 in PBS containing 1% normal goat serum at 48C for 24 h. After being rinsed in PBS, the sections were incubated with Texas-red conjugated goat anti-mouse IgG ŽJackson. at a dilution of 1:100
Tissues were immersed in a solution of 2% glutaraldehyde in 0.1 M Sorensen’s phosphate buffer ŽpH 7.4. for 1 ¨ h at 48C. After being rinsed in phosphate buffer several times for 30 min, the specimens were postfixed in 2% osmium tetroxide in 0.1 M Sorensen’s phosphate buffer ¨ ŽpH 7.4. for 30 min at 48C. They were then dehydrated in a graded series of ethyl alcohols, immersed in propylene oxide for 30 min at room temperature and finally embedded in Epon. The specimens were cut into semithin sections Ž0.3–0.5 m m. which were stained with Azure blue, and ultrathin sections, which were contrasted with uranyl acetate and lead citrate. The semithin sections were examined in a photonic microscope ŽAristoplan, Leitz. and the ultrathin sections in an electron microscope ŽPhilips EM 400T..
3. Results 3.1. Light microscopy The DMP of the pig duodenum was located between the inner and outer circular muscle layer ŽFig. 1A, B.. The inner circular muscle layer was a very thin layer containing 2–3 strata of smooth muscle cells. These cells were smaller and denser than those of the outer circular layer, which was about ten times thicker. The DMP consisted of a cluster of non-neuronal cells and nerve fibres, connected together by networks consisting mainly of nerve fibres and a few rare non-neuronal cells. When cutting sections, one therefore crossed at random through either the tracts ŽFig. 1A. or the cell clusters ŽFig. 1B..
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Fig. 1. Photomicrographs of semithin sections of the pig duodenum. The deep muscular plexus was composed of clusters of non-neuronal cells ŽB, arrows. linked together by thin tracts ŽA, arrows.. This plexus was located between the outer ŽOCM. and inner ŽICM. circular muscle layer. The inner circular muscle layer was separated from the external submucous plexus ŽESP. by loose connective tissue. The outer circular muscle layer was separated from the longitudinal muscle layer ŽLM. by the myenteric nervous plexus ŽMP..
3.2. Immunohistochemistry
3.3. Electron microscopy
In all the sections examined, the synaptophysin-like immunoreactivity was located in the ganglionated plexus, myenteric and external submucous plexus, the varicose nerve fibres present in the outer circular muscle layer and in the DMP; no immunoreactivity was revealed in the inner circular muscle layer ŽFig. 2A.. In order to illustrate the density of the immunoreactive material shown in Fig. 2A, the data in this figure were digitized in order to obtain a vertical picture of the grey level histograms ŽFig. 2B.. The pattern of distribution of the grey level histograms indicated that the density of the synaptophysin-like immunoreactivity was particularly high in the ganglionated plexus and the DMP.
Our ultrastructural study on the pig DMP showed the presence of various nerve fibres, Schwann cells, macrophages and interstitial cells. 3.3.1. Schwann cells The Schwann cells were sometimes surrounded by non-myelinated nerve fibres ŽFig. 3A.. They had a central nucleus containing both marginal and perinucleolar heterochromatin ŽFig. 3A.. Their cytoplasm formed a thin perinuclear rim with long extensions. The perinuclear cytoplasm contained numerous ribosomes, a few mitochondria, a few endoplasmic rough reticulum, one centriole, a poorly
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Fig. 2. Synaptophysin-like immunoreactivity in the pig duodenum. ŽA. A conventional fluorescence photomicrograph of the synaptophysin immunoreactivity present in varicose nerve fibres distributed throughout the duodenal wall is shown. It can be noted that the density of the immunoreactivity was particularly high in the myenteric Žmp. and deep muscular plexus Žarrows.. ŽB. A vertical picture of the grey level histograms of the synaptophysin immunoreactivity given in ŽA. is shown. This picture was obtained by digitalizing of the photomicrograph in ŽA.; the grey level histograms are indicated by means of artificial colors. LM: longitudinal muscle; OCM: outer circular muscle; ICM: inner circular muscle; SM: submucosae; mp: myenteric plexus; esp: external submucous plexus.
developed Golgi apparatus and some rare lysosomes. The cytoplasmic extensions enveloped numerous nerve fibres. Microtubules and gliofilaments were present throughout the cytoplasm. The Schwann cells were always surrounded by a continuous thick basal lamina.
3.3.2. NerÕe fibres The nerve fibres contained vesicles of several kinds: either small clear synaptic vesicles ŽFig. 4B., or clusters of small dense vesicles ŽFig. 3C., or more frequently heterogeneous populations including dense-cored vesicles, small
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Fig. 3. Electron photomicrograph of Schwann cells ŽSC. of the deep muscular plexus of the pig duodenum ŽA.; these cells were located inside the deep muscular plexus located between the inner ŽICM. and the outer ŽOCM. circular muscle layers. The Schwann cells were delimited by a continuous basal lamina ŽBL. closely surrounding the nerve fibres ŽNF.. Electron photomicrographs of nerve fibres ŽNF. of the deep muscular plexus of the pig duodenum ŽB and C.. ŽB. A nerve fibre in contact with a fibroblastic-like cell ŽFLC. is shown; note the presence in the nerve fibre of small clear vesicles Žsmall arrows., large dense-cored vesicles Žlarge arrow., small areolated vesicles Žhead arrows. and large granular vesicles Žstar.. ŽC. One of the nerve fibres, facing the inner circular muscle layer ŽICM. contained similar numerous dense-cored vesicles, while in the other fibre, different types of vesicles were observed. Scale bar: 0.4 m m.
clear vesicles, small areolated vesicles and large granular vesicles ŽFig. 3B.. All these nerve fibres were arranged parallel to the adjacent smooth muscle cells and were often closely apposed to the two types of ICs described below.
3.3.3. Interstitial cells The ICs present in the DMP were of two kinds, namely interstitial fibroblastic-like cells ŽFLC. ŽFig. 4A. and interstitial dense cells ŽIDC. ŽFig. 4B..
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Fig. 4. Electron photomicrographs of a fibroblastic-like cell and interstitial dense cell of the deep muscular plexus of the pig duodenum. ŽA. Note that the fibroblastic-like cell ŽFLC. was apposed to a nerve fibre ŽNF., both of which were delimited by the inner ŽICM. and outer ŽOCM. circular muscle layers; ŽB. An interstitial dense cell ŽIDC. characterized by a dense heterochromatin ŽH. nucleus ŽN. with a voluminous nucleolus ŽNu., a cytoplasm containing numerous ribosomes and caveolae Žarrows. is shown; note the presence of a nerve fibre ŽNF. containing numerous small clear vesicles, closely apposed to the interstitial cell. Scale bar: 0.4 m m.
The FLC always ran parallel to the smooth muscle cells and were electron-clear and bipolar ŽFig. 4A, Fig. 5A.. They had a central ovoid nucleus with an irregular contour, which was rimmed by a fairly regular heterochromatin border and contained a voluminous nucleolus ŽFig. 4A..
The FLC were characterized by an abundant endoplasmic rough reticulum containing a medium electron-dense fibrillar component, and numerous mitochondria ŽFig. 5A.. Islets consisting of smooth endoplasmic reticulum tubules of dense material were also present ŽFig. 5B.. In some
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Fig. 5. Electron photomicrographs of fibroblastic-like cells ŽFLC. of the deep muscular plexus of the pig duodenum, interposed between the inner ŽICM. and outer ŽOCM. circular muscle layer. In the FLC, note the presence of abundant endoplasmic rough reticulum ŽE. surrounding mitochondria ŽM., tubules of smooth endoplasmic reticulum ŽSER., lysosomes ŽL. and ribosomes ŽR.. Scale bar: 0.4 m m.
places, voluminous secondary lysosomes had accumulated, as well as numerous free ribosomes ŽFig. 5C.. In the vicinity of the nucleus, the Golgi apparatus could be observed ŽFig. 6A.. The latter was characterized by the presence of a few sacculae and a very large number of vesicles, some of which were coated vesicles. A cilium
was sometimes observed in the vicinity of the Golgi apparatus ŽFig. 6A.. The FLC were found to be the sites of intensive pinocytic activity ŽFig. 6A.. The FLC were frequently connected together by interdigitations ŽFig. 6B.. They did not contain any basal lamina and were much more numerous than the IDC. The FLC were interposed
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Fig. 6. Electron photomicrographs of fibroblastic-like cells ŽFLC. of the deep muscular plexus of the pig duodenum. The FLC were characterized by a Golgi apparatus ŽG. close to the nucleus, a pinocytic ŽA, arrow. activity and a cilium ŽC.. They were connected together by interdigitations ŽB, arrow., desmosomes ŽB, head arrows, and E, arrow. and gap junction ŽC, arrow.; they were in close position with nerve fibers ŽNF.Ž6D.. Scale bar: 0.4 m m.
between nerve endings and smooth muscle cells belonging to either the inner or outer circular muscle layer. The nerve endings were linked to the FLC by membrane apposition only ŽFig. 6D., whereas the FLC were connected to each other and to smooth muscle cells by desmosomes ŽFig. 6B, E. and more rarely by gap junctions ŽFig. 6C.; among 50
sections from 50 different FLC, only two gap junctions were identified, while 30 desmomes were observed. The IDC were either stellate ŽFig. 7A. or bipolar ŽFig. 7B, C. and ran parallel to the smooth muscle cells ŽFig. 4B, Fig. 7B, C.. They had a multi-lobed nucleus which was characterized by large amounts of marginal and peri
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Fig. 7. Electron photomicrographs of interstitial cells of the deep muscular plexus of the pig duodenum. The interstitial dense cells ŽIDC. were located between the inner ŽICM. and outer ŽOCM. circular muscle layer. They were often close to the fibroblastic-like cells ŽFLC.. Note that the IDC were interposed between a nerve fibre ŽNF. and a smooth muscle cell ŽMC.. The IDC were characterized by a discontinuous basal lamina ŽFig. 7, arrows., large mitochondria ŽM. ŽA and B., a Golgi apparatus ŽG. ŽA., an endoplasmic rough reticulum ŽE. which was often dilated ŽA and C., numerous ribosomes ŽR. ŽC., polysomes ŽP. ŽB., and dense bodies ŽC, arrows.. Note that IDC were connected to the adjacent smooth muscle cell by small gap junctions ŽD, arrows.. Scale bar: 0.4 m m.
nucleolar heterochromatin ŽFig. 4B, Fig. 7B, C. and a voluminous nucleolus ŽFig. 4B.. The cytoplasm was found to contain a few large mitochondria, numerous ribosomes and polysomes ŽFig. 4B, Fig. 7B, C., endoplasmic rough
reticulum with some dilated cisternae ŽFig. 7A, C., and a Golgi apparatus ŽFig. 7A.. The IDC were furthermore characterized by the presence of dense bodies ŽFig. 7C., numerous caveolae ŽFig. 4B, Fig. 7B, D., a discontinuous
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basal lamina ŽFig. 7A., and the occurrence of pinocytic activity ŽFig. 7B.. The IDC were interposed between nerve fibres to which they were closely apposed ŽFig. 4B, Fig. 7A. and smooth muscle cells to which they were connected by gap junctions ŽFig. 7D.. The presence of ICs was furthermore observed in the interlamellar septa, in the aganglionic tracts of the outer circular muscle layer, and in the plexus muscularis superficialis. The FLC predominated in the DMP and the external circular muscle layer, and the IDC in the septa and the plexus muscularis superficialis. No nerve fibres or ICs were detected in the inner circular muscle layer.
4. Discussion To our knowledge, this is the first detailed ultrastructural study to be carried out so far on the deep muscular plexus of the pig duodenum. The main histological features of the pig duodenal DMP observed in the present study were broadly similar to those of the small intestine of the other species studied so far, namely the guinea-pig ŽGabella, 1972; Wilson et al., 1987; Zhou and Komuro, 1992a,b., bat ŽYamamoto, 1977., rat ŽKomuro and Seki, 1995., mouse ŽRumessen et al., 1982; Yamamoto, 1977., dog ŽTorihashi et al., 1993., and Man ŽFaussone-Pellegrini and Cortesini, 1983.. In addition, these histological features of small intestine DMP are also similar to those of the plexus entericus extremus described in the colon of the cat ŽConklin and Du, 1990., dog ŽLiu et al., 1994; Serio et al., 1991. and Man ŽFaussone-Pellegrini, 1992; Faussone-Pellegrini et al., 1990.. The main cellular components of the pig duodenal DMP were found to belong to the same broad categories of cells as those described in other species, since they consist of macrophages, Schwann cells, nerve fibres and ICs. In the present study, the analysis of synaptophysin-like immunoreactivity clearly showed that a large number of varicose nerve fibres present in the DMP contained small clear vesicles. The existence of these vesicles was further confirmed at the ultrastructural level. Our ultrastructural results show, however, that other types of vesicles are often co-localized with these small clear vesicles. In the DMP of the guinea-pig, the small clear vesicles contain either VIP or SP, sometimes along with large granular enkephalin-containing vesicles; VIP and SP may be involved in the transmission of inhibitory and excitatory inputs, respectively to smooth muscle, whereas enkephalins may modulate both excitatory and inhibitory nerve fiber transmission ŽLlewellyn-Smith et al., 1988.. Pig myenteric neurons were also found to contain VIP, SP and enkephalins as well as galanin, CGRP, somatostatin, NPY and serotonin ŽTimmermans et al., 1991.. The presence of enkephalin-like immunoreactive fibres has been previously demonstrated in the pig DMP ŽHenry et al., 1996.. It has been suggested that the DMP nerves in the pig may
correspond to the Dogiel’s type II neurons located in the myenteric plexus ŽScheuermann et al., 1989.. It is therefore possible that transmitters present in nerve fibres of the pig DMP may have similar subcellular localizations and functions to those described in the guinea-pig ŽLlewellynSmith et al., 1988.. The lack of any anatomical connecti vity between the DMP and the external submucosal plexus observed in this study strongly suggests that all the DMP nerve fibres may originate from the myenteric plexus as previously found to occur in the guinea-pig ŽWilson et al., 1987.. It is worth noting that a number of nerve strands run from the myenteric plexus to the external submucous plexus ŽScheuermann et al., 1987b; unpublished data.. This pattern of organization of the intramural pathways strongly suggests the existence of peripheral reflex loops between the external submucous plexus, the myenteric plexus and the DMP. The results of the present study show that in the pig DMP the nerve fibres run between ICs of two main types: those of the FLC type, which predominated, and those of the IDC type. This overall picture suggests that FLC may therefore be ‘interstitial cells’, which were defined in the review by Komuro Ž1990. as originating from fibroblasts but being differentiated depending on the neighbouring structures, nerves and muscles. The FLC present in the pig DMP were similar to those of the rat DMP, as well as to the ZIO positive ICs observed in the guinea-pig DMP ŽZhou and Komuro, 1992a,b, 1995.. The present results indicate however that in the pig DMP, the FLC never contain any of the basal lamina or glycogen particles present in the other group of FLC described in the guineapig DMP ŽZhou and Komuro, 1992a,b, 1995.. The existence of a cilium has also been described in mouse FLC, but its functional role still remains to be determined ŽRumessen et al., 1982.. The main characteristics of the IDC observed here in the pig DMP indicate that they are of the myoid-like type and tend to resemble the ICs described in the lovebird ŽImaizumi and Hama, 1969., dog ŽDuchon et al., 1974; Torihashi et al., 1993., bat ŽYamamoto, 1977., mouse ŽRumessen et al., 1982; Yamamoto, 1977., rat ŽKomuro and Seki, 1995., guinea-pig ŽZhou and Komuro, 1992b., and Man ŽFaussone-Pellegrini, 1983. where they are the only type of ICs present. The pig ICs resemble those described in Man, however, in that they contain only very few small gap junctions, whereas the ICs of the other species listed above contain a large number of long gap junctions. The IDC might correspond to a non-differen tiated or immature type of smooth muscle cell ŽYamamoto, 1977.. Both types of ICs detected here in the pig DMP were closely apposed to nerve fibres. Gabella Ž1972. has reported that the close appositions between ICs and nerves are actually connections which may mediate communications. In addition, we observed that the ICs in the pig DMP were connected by gap junctions not only to each other,
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but also to smooth muscle cells of both the inner and outer circular muscle layers. Although these gap junctions were small and sparse, the finding that connexin mRNA is present in the dog colon ŽLi et al., 1993. suggests that there may exist minute gap junctions which are not detectable at the electron microscopic level. Finally, the interstitial cells of Cajal are a population of cells in the gastrointestinal tract which play a role in the control of gut motility ŽHagger et al., 1997.; these cells are characterized by their appearance, which is either bipolar or stellate, their network arrangement, and by the fact that they are interposed between nerve fibres and smooth muscle cells. Both types of ICs cells described here in the pig DMP can therefore be said to be interstitial cells of Cajal. In this study, no innervation was detected in the inner circular muscle layer of the pig duodenum. In view of the particular smooth muscle cells of which it is composed, this layer probably has a specific function, as previously suggested ŽGabella, 1972.. In the pig, the inner circular muscle layer was contacted only by ICs of the DMP which were themselves connected to nerve fibres. Some authors ŽGabella, 1972. have concluded that the DMP may control only the inner circular muscle layer, while others ŽDuchon et al., 1974. favour the idea that it may control only the outer circular layer. The present results obtained on the pig suggest that the DMP may be involved in the control of both inner and outer circular muscle layers via the ICs. Finally, the data obtained on various species, including pigs, suggest that the role of the DMP in the control of the circular muscle layer probably differs from one species to another. The network of ICs in the pig DMP, which are interposed between nerves and smooth muscle cells, differ from the ICC–AP located in the myenteric plexus. In the pig Žunpublished data. as in the guinea-pig ŽZhou and Komuro, 1995. and Man ŽFaussone-Pellegrini et al., 1990., the ICC–AP constitute an independent cellular network, having only rare, if any, contacts with the nervous pathways. Moreover, in the small intestine of c-kit protooncogene mutant mice, it has been reported that the number of ICC–AP was considerably lower than in normal mice, while the smooth muscle electrical slow waves were completely absent ŽWard et al., 1994.. The mutation had no effect however on either the ICC–DMP or the excitatory and inhibitory nervous inputs to the smooth muscle cells. It has been suggested that in the small intestine of the mouse, the ICC–AP may be mainly involved in the pacemaker activity of smooth muscle cells ŽWard et al., 1994., while the ICC–DMP may mediate neural inputs ŽBurns et al., 1996.. The present finding that the ICs ŽFLC and IDC. of the pig DMP are interposed between nerve fibres and smooth muscle cells is consistent with the intercalation hypothesis first put forward by Cajal Ž1911., who suggested that these cells might play a fundamental role in neurotransmission. The ICs of the pig DMP may therefore be involved at least
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in the integration and conduction of excitatory and inhibitory nervous inputs from myenteric motoneurons to the inner and outer circular muscle layers. The clear-cut distinction found to exist in the present study between the two types of ICs Žfibroblastic- and myoid-like. present in the pig DMP indicates that the ICs of each type may also be involved in some other specific activity, which still remains to be determined. Acknowledgements The authors wish to express their sincere thanks to Christine Meyer and Jean Claude Stamegna for their excellent technical assistance. The authors would also like to thank Pierre Rage for his help with the computer image analysis. References Burns, A.J., Lomax, A.E.J., Torihashi, S., Sanders, K.M., Ward, S.M., 1996. Interstitial cells of Cajal mediate inhibitory transmission in the stomach. Proc. Natl. Acad. Sci. USA 93, 12008–12013. Cajal, S.R., 1893. Sur les ganglions et plexus nerveux de l’intestin. Comp. Rend. Sean. ´ Soc. Biol. 5, 217–223. Cajal, S.R., 1911. Histologie du systeme ` nerveux de l’Homme et des vertebres. ´ ´ Maloine ŽParis. 2, 891–942. Conklin, J.L., Du, C., 1990. Pathways of slow-wave propagation in proximal colon of cats. Am. J. Physiol. 258, G894–G903. Duchon, G.R., Henderson, E., Daniel, E., 1974. Circular muscle layers in the small intestine. In: Daniel, E. ŽEd.., Proceedings of the Fourth International Symposium on Gastrointestinal Motility, Mitchell Press, Vancouver, pp. 636–646. Faussone-Pellegrini, M.S., 1982. Ultrastuctura e topografia delle cellule interstiziali del Cajal nello strato circolare della tunica muscolare del ileo e del colon del ratto. Bol. Soc. Ital. Biol. Sper. 58, 1260–1265. Faussone-Pellegrini, M.S., 1983. Le caracteristiche ultrastructurali della tunica muscolare del ileo e del colon di ratto: A. Porzione interna della strato circolare. Arch. Ital. Anat. Embryol. 88, 25–40. Faussone-Pellegrini, M.S., 1984. 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 E.M. study. Anat. Embryol. ŽBerlin. 169, 151–158. Faussone-Pellegrini, M.S., 1992. Histogenesis, structure and relationships of interstitial cells of Cajal: from morphology to functional interpretation. Eur. J. Morphol. 30, 137–148. Faussone-Pellegrini, M.S., Cortesini, C., 1983. Some ultrastructural features of the muscular coat of human small intestine. Acta Anat. ŽBasel. 115, 47–68. Faussone-Pellegrini, M.S., Pantalone, D., Cortesini, C., 1990. Smooth muscle cells, interstitial cells of Cajal and myenteric plexus interrelationships in the human colon. Acta Anat. ŽBasel. 139, 31–44. Gabella, G., 1972. Innervation of the intestinal muscular coat. J. Neurocytol. 1, 341–362. Gunn, M., 1968. Histological and histochemical observations on the myenteric and submucous plexuses of mammals. J. Anat. 102, 223– 239. Hagger, R., Finlayson, C., Jeffrey, I., Kumar, D., 1997. Role of the interstitial cells of Cajal in the control of gut motility. Br. J. Surg. 84, 445–450. Hara, Y., Kubota, M., Szurszewski, J.H., 1986. Electrophysiology of smooth muscle of the small intestine of some mammals. J. Physiol. 372, 501–520.
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