Spatial relationship between telocytes,interstitial cells of Cajal and the enteric nervous system in the human ileum and colon

Telocytes (TCs) are recently described interstitial cells, present in almost all human organs. Among many other functions, TCs regulate gastrointestinal motility together with the interstitial cells of Cajal (ICCs). TCs and ICCs have close localization in the human myenteric plexus; however, the exact spatial relationship cannot be clearly examined by previously applied double immunofluorescence/confocal microscopy. Data on TCs and submucosal ganglia and their relationship to intestinal nerves are scarce. The aim of the study was to analyse the spatial relationship among these components in the normal human ileum and colon with double CD34/CD117 and CD34/S100 immunohistochemistry and high-resolution light microscopy. TCs were found to almost completely encompass both myenteric and submucosal ganglia in ileum and colon. An incomplete monolayer of ICCs was localized between the TCs and the longitudinal muscle cells in ileum, whereas only scattered ICCs were present on both surfaces of the colonic myenteric ganglia. TC-telopodes were observed within colonic myenteric ganglia. TCs, but no ICCs, were present within and around the interganglionic nerve fascicles, submucosal nerves and mesenterial nerves, but were only observed along small nerves intramuscularly. These anatomic differences probably reflect the various roles of TCs and ICCs in the bowel function.     

PKC-epsilon translocation in enteric neurons and interstitial cells of Cajal in response to muscarinic stimulation

Interstitial cells of Cajal in the deep muscular plexus (ICC-DMP) of the small intestine express excitatory neurotransmitter receptors. We tested whether ICC-DMP are functionally innervated by cholinergic neurons in the murine intestine. Muscles were stimulated by intrinsic nerves and ACh and processed for immunohistochemistry to determine these effects on PKC-epsilon activation. Under control conditions, PKC-epsilon-like immunoreactivy (PKC-epsilon-LI) was only observed in myenteric neurons within the tunica muscularis. Electrical field stimulation or ACh caused translocation of neural PKC-epsilon-LI from the cytosol to a peripheral compartment. After stimulation, PKC-epsilon-LI was found in spindle-shaped cells in the DMP. These cells were identified as ICC-DMP by Kit-LI and vimentin-LI. PKC-epsilon-LI in ICC-DMP and translocation of PKC epsilon-LI in neurons were blocked by tetrodotoxin or atropine, suggesting that these responses were due to activation of muscarinic receptors. Western blots also confirmed translocation of PKC-epsilon-LI. In conclusion, PKC-epsilon translocation is linked to muscarinic receptor activation in ICC-DMP and a subpopulation of myenteric neurons. These studies demonstrate that ICC-DMP are functionally innervated by excitatory motoneurons.     

Relationship between interstitial cells of Cajal, fibroblast-like cells and inhibitory motor nerves in the internal anal sphincter

Interstitial cells of Cajal (ICC) have been shown to participate in nitrergic neurotransmission in various regions of the gastrointestinal (GI) tract. Recently, fibroblast-like cells, which are positive for platelet-derived growth factor receptor ?? (PDGFR??⁺), have been suggested to participate additionally in inhibitory neurotransmission in the GI tract. The distribution of ICC and PDGFR??⁺ cell populations and their relationship to inhibitory nerves within the mouse internal anal sphincter (IAS) are unknown. Immunohistochemical techniques and confocal microscopy were therefore used to examine the density and arrangement of ICC, PDGFR??⁺ cells and neuronal nitric-oxide-synthase-positive (nNOS⁺) nerve fibers in the IAS of wild-type (WT) and W/W ^v mice. Of the total tissue volume within the IAS circular muscle layer, 18% consisted in highly branched PDGFR??⁺ cells (PDGFR??⁺-IM). Other populations of PDGFR??⁺ cells were observed within the submucosa and along the serosal and myenteric surfaces. Spindle-shaped intramuscular ICC (ICC-IM) were present in the WT mouse IAS but were largely absent from the W/W ^v IAS. The ICC-IM volume (5% of tissue volume) in the WT mouse IAS was significantly smaller than that of PDGFR??⁺-IM. Stellate-shaped submucosal ICC (ICC-SM) were observed in the WT and W/W ^v IAS. Minimum surface distance analysis revealed that nNOS⁺ nerve fibers were closely aligned with both ICC-IM and PDGFR??⁺-IM. An even closer association was seen between ICC-IM and PDGFR??⁺-IM. Thus, a close morphological arrangement exists between inhibitory motor neurons, ICC-IM and PDGFR??⁺-IM suggesting that some functional interaction occurs between them contributing to inhibitory neurotransmission in the IAS.     

Intimate relationship between interstitial cells of Cajal and enteric nerves in the guinea-pig small intestine

Recent studies have suggested that enteric inhibitory neurotransmission is mediated via interstitial cells of Cajal in some gastrointestinal tissues. This study describes the physical relationships between enteric neurons and interstitial cells of Cajal in the deep muscular plexus (IC-DMP) of the guinea-pig small intestine. c-Kit and vimentin were colocalized in the cell bodies and fine cellular processes of interstitial cells of the deep muscular plexus. Anti-vimentin antibodies were subsequently used to examine the relationships of interstitial cells with inhibitory motor neurons (as identified by nitric oxide synthase-like immunoreactivity) and excitatory motor neurons (using substance P-like immunoreactivity). Neurons with nitric oxide synthase- and substance P-like immunoreactivities were closely associated with the cell bodies of interstitial cells and ramified along their processes for distances greater than 300 7m. With transmission electron microscopy, we noted close relationships between interstitial cells and the nitric oxide synthase- and substance P-like immunoreactive axonal varicosities. Varicosities of nitric oxide synthase and substance P neurons were found as close as 20 and 25 nm from interstitial cells, respectively. Specialized junctions with increased electron density of pre- and postsynaptic membranes were observed at close contact points between nitric oxide synthase- and substance P-like immunoreactive neurons and interstitial cells. Close structural relationships (approximately 25 nm) were also occasionally observed between either nitric oxide synthase- and substance P-like immunoreactive varicosities and smooth muscle cells of the outer circular muscle layer. The data suggest that interstitial cells in the deep muscle plexus are heavily innervated by excitatory and inhibitory enteric motor neurons. Thus, these interstitial cells may provide an important, but probably not exclusive, pathway for nerve-muscle communication in the small intestine.     

Enteric motor neurons form synaptic-like junctions with interstitial cells of Cajal in the canine gastric antrum

Morphological studies have shown synaptic-like structures between enteric nerve terminals and interstitial cells of Cajal (ICC) in mouse and guinea pig gastrointestinal tracts. Functional studies of mice lacking certain classes of ICC have also suggested that ICC mediate enteric motor neurotransmission. We have performed morphological experiments to determine the relationship between enteric nerves and ICC in the canine gastric antrum with the hypothesis that conservation of morphological features may indicate similar functional roles for ICC in mice and thicker-walled gastrointestinal organs of larger mammals. Four classes of ICC were identified based on anatomical location within the tunica muscularis. ICC in the myenteric plexus region (IC-MY) formed a network of cells that were interconnected to each other and to smooth muscle cells by gap junctions. Intramuscular interstitial cells (IC-IM) were found in muscle bundles of the circular and longitudinal layers. ICC were located along septa (IC-SEP) that separated the circular muscle into bundles and were also located along the submucosal surface of the circular muscle layer (IC-SM). Immunohistochemistry revealed close physical associations between excitatory and inhibitory nerve fibers and ICC. These contacts were synaptic-like with pre- and postjunctional electron-dense regions. Synaptic-like contacts between enteric neurons and smooth muscle cells were never observed. Innervated ICC formed gap junctions with neighboring smooth muscle cells. These data show that ICC in the canine stomach are innervated by enteric neurons and express similar structural features to innervated ICC in the murine GI tract. This morphology implies similar functional roles for ICC in this species.     

Ultrastructural characterization of interstitial cells of Cajal associated with the submucosal plexus in the proximal colon of the guinea pig

Interstitial cells of Cajal (ICC) associated with the submucosal (submucous) plexus (ICC-SP) in the proximal colon of the guinea pig were studied by immunohistochemistry and electron microscopy. Whole-mount stretch preparations with c-Kit immunohistochemistry revealed that a number of ICC-SP constituted a dense cellular network around the submucosal plexus. Some of these ICC-SP were observed in the vicinity of the muscularis mucosae in sections immunostained for c-Kit and α-smooth muscle actin. Ultrastructural observation demonstrated, for the first time, that ICC-SP of the proximal colon of the guinea pig retained typical ultrastructural characteristics of ICC repeatedly reported in association with the tunica muscularis of the gastrointestinal tract: a basal lamina, caveolae, many mitochondria, abundant intermediate filaments and the formation of gap junctions with the same type of cells. The most remarkable ultrastructural finding was the presence of thick bundles composed of the processes of ICC-SP connected to each other via large gap junctions. These ICC-SP might be involved in the main mucosal functions of the proximal colon of the guinea pig, namely the transportation of water and electrolytes, possibly via their involvement in the spontaneous contractions of the muscularis mucosae.     

Pacemaker potentials generated by interstitial cells of Cajal in the murine intestine

Pacemaker potentials were recorded in situ from myenteric interstitial cells of Cajal (ICC-MY) in the murine small intestine. The nature of the two components of pacemaker potentials (upstroke and plateau) were investigated and compared with slow waves recorded from circular muscle cells. Pacemaker potentials and slow waves were not blocked by nifedipine (3 µM). In the presence of nifedipine, mibefradil, a voltage-dependent Ca²⁺ channel blocker, reduced the amplitude, frequency, and rate of rise of upstroke depolarization (dV/dt_max) of pacemaker potentials and slow waves in a dose-dependent manner (1–30 µM). Mibefradil (30 µM) changed the pattern of pacemaker potentials from rapidly rising, high-frequency events to slowly depolarizing, low-frequency events with considerable membrane noise (unitary potentials) between pacemaker potentials. Caffeine (3 mM) abolished pacemaker potentials in the presence of mibefradil. Pinacidil (10 µM), an ATP-sensitive K⁺ channel opener, hyperpolarized ICC-MY and increased the amplitude and dV/dt_max without affecting frequency. Pinacidil hyperpolarized smooth muscle cells and attenuated the amplitude and dV/dt_max of slow waves without affecting frequency. The effects of pinacidil were blocked by glibenclamide (10 µM). These data suggest that slow waves are electrotonic potentials driven by pacemaker potentials. The upstroke component of pacemaker potentials is due to activation of dihydropyridine-resistant Ca²⁺ channels, and this depolarization entrains pacemaker activity to create the plateau potential. The plateau potential may be due to summation of unitary potentials generated by individual or small groups of pacemaker units in ICC-MY. Entrainment of unitary potentials appears to depend on Ca²⁺ entry during upstroke depolarization. pacemaker activity; slow waves; gastrointestinal motility; calcium channel     

Visualization of interstitial cells of Cajal in the mouse colon by vital staining

Interstitial cells of Cajal (ICCs) are believed to be a major element in generating the spontaneous rhythm of the gastrointestinal tract. A prominent problem in the study of these cells has been the difficulty in observing them in intact tissues. We used the lipophilic dye DiI to stain ICCs in the submucosal-circular muscle border of freshly dissected mouse colon. The placement of small DiI crystals in this area resulted in the labeling of ICC-like cells. Two main morphological cell types, viz., bipolar and multipolar, were noted. Bipolar cells had two primary processes emerging from the poles of an elongated soma. The mean length of these processes was 78.7 μm. These cells constituted 42.3% of the sample (n=105). Multipolar cells (54.3% of total) had a less elongated soma and extended 3–6 main processes whose mean length was 56.3 μm. These processes showed no preferred direction. The length of the primary processes of bipolar cells was 40% greater than that of multipolar cells (P<0.02). Three cells (2.9%) had only one primary process. The DiI stain could be converted into a stable electron-opaque product. Electron-microscopic observations showed that these cells had the typical appearance of ICCs reported in previous studies. This staining method should be useful for physiological investigations of ICCs in gastrointestinal tissues. Received: 16 September 1997 / Accepted: 11 November 1997     

Ultrastructure of interstitial cells of Cajal in myenteric plexus of human colon

The role of the interstitial cells of Cajal (ICC) associated with the myenteric plexus (ICC-MP) as regulators of the motility of the colonic external muscle remains unclear. Ultrastructural studies of myenteric interstitial cells are lacking in human colon. We therefore characterized the distinctive ultrastructure of these cells in the myenteric region of the colon by transmission electron microscopy of the region between the main muscle layers in all parts of the colon in unaffected areas of resected specimens from nine adult human patients. ICC-MP were similar in various colonic regions and had myoid features such as scattered caveolae, prominent intermediate filaments, and cytoplasmic dense bodies. We found characteristic dense membrane-associated bands with a patchy basal lamina, invaginating cellular protrusions (peg and socket junctions) between ICC and between ICC and muscle cells, and close contacts (<100 nm) between ICC and nerves. No gap junctions were observed. Fibroblast-like cells (FLC) were abundant showing well-developed secretory organelles, including coated vesicles, but lacked prominent intermediate filaments and caveolae. FLC had a patchy basal lamina, and peg and socket junctions were observed between them. Macrophage-like cells frequently occurred in close apposition with FLC and, more seldomly, with ICC-MP. The ultrastructure of ICC and FLC in the myenteric region of the human colon thus differs characteristically, but significant overlaps in the ultrastructure between ICC and FLC might complicate any interpretation in pathological ultrastructural studies of the human colonic muscle layer. An erratum to this article can be found at     

Characterization of interstitial cells of Cajal in the subserosal layer of the guinea-pig colon

Interstitial cells of Cajal in the subserosa (ICC-SS) of the guinea-pig proximal colon were studied by immunohistochemistry for c-Kit receptors and by transmission electron microscopy. These cells were distributed within a thin layer of connective tissue space immediately beneath the mesothelium and were multipolar with about five primary cytoplasmic processes that divided further into secondary and tertiary processes to form a two-dimensional network. Ultrastructural observations revealed that ICC-SS were connected to each other via gap junctions. They also formed close contacts and peg-and-socket junctions with smooth muscle cells. Three-dimensional analysis of confocal micrographs revealed that the cytoplasmic processes of ICC-SS had contacts with interstitial cells in the longitudinal muscle layer. Taking account of the location and peculiar arrangement of the ICC-SS and the main functions of the proximal colon, i.e. the absorption and transport of fluids, we suggest that the superficial network of ICC-SS acts as a stretch receptor to detect circumferential expansion and swelling of the colon wall and triggers the contraction of the longitudinal muscle to accelerate the drainage of fluids from the colon.     

Smooth muscle cells, interstitial cells of Cajal and myenteric plexus interrelationships in the human colon

The plane between longitudinal and circular muscle of human colon, as revealed on examination with light and electron microscopes, has no clear-cut border. Some groups of smooth muscle cells, obliquely oriented and with features similar to both circular and longitudinal ones--the connecting muscle bundles--run from one muscle layer to another. Other groups of smooth muscle cells, possessing their own specific ultrastructural features--the myenteric muscle sheaths--, make up envelopes of variable thickness around some myenteric ganglia and nerve strands, partially or completely embedding them in one or other muscle layer. Non-neuronal, non-muscular cells (interstitial cells of Cajal, covering cells, fibroblast-like and macrophage-like cells) complicate the texture of the myenteric muscle sheaths, creating an intricate, interconnected cellular network inside them, widespread among nerve bundles and smooth muscle cells; however, only interstitial cells have cell-to-cell junctions also with the smooth muscle cells and nerve endings. These data document the existence in this colonic area of two different types of muscle cell arrangements, one of which, the myenteric muscle sheath, only contains putative pacemaker cells.     

Plasticity of interstitial cells of Cajal: a study of mouse colon

Ablation of the myenteric plexus in mouse colon with the detergent benzalkonium chloride (BAC) is followed by considerable recovery of the nerves, indicating that this plexus is capable of regeneration and has plasticity. Interstitial cells of Cajal (ICC) are closely associated with enteric nerves, and the acquisition and maintenance of their adult phenotype are nerve-dependent. Little is known about the regenerative processes of ICC or about the possible dependence of these processes on neurons. To address these questions, we ablated the myenteric plexus in the mouse colon with BAC and followed changes in the adjacent ICC (ICC-MP) from day 2 to day 70 after treatment, by using c-kit-immunohistochemistry and electron microscopy. In the untreated area, c-kit-positive cells and ICC-MP with normal ultrastructural features were always present. The region partially affected by BAC contained some c-kit-positive cells, and either normal or vacuolated ICC-MP were observed by electron microscopy. Moreover, at days 60–70, ICC-MP with particularly extended rough endoplasmic reticulum were present in this area. In the treated area, either denervated or reinnervated, c-kit-positive cells were always absent. By day 14 after BAC treatment, nerve fibers had started to grow back into the treated region and, in the reinnervated area, cells with fibroblast-like features appeared and were seen to contact both nerve endings and smooth muscle cells and to acquire some typical ICC features. Thus, ICC are vulnerable to external insult but appear to have some ability to regenerate.This work was supported by the US-Israel Binational Science Foundation (BSF, 98-00185; to M.H.) and University funds “quota di ateneo ex 60%” (M.-S. F.-P.).     

Distribution, development and proliferation of interstitial cells of Cajal in murine colon: an immunohistochemical study from neonatal to adult life

This paper aimed at investigating the alterations in interstitial cells of Cajal (ICC) in the proximal, middle and distal colon of mice from 0-day to 56-day post-partum (P0–P56) by immunohistochemistry. The Kit⁺ ICC, which situated around myenteric nerve plexus (ICC–MY) were prominent at birth, meanwhile those cells within the smooth muscle layers (ICC–IM) and in the connective tissue beneath serosa (ICC–SS) began to appear. ICC–SM, which located at the submucosal border of circular muscle layer emerged at P6 in the proximal colon and subsequently in the distal colon at P8, and ICC in the oral side of colon revealed an earlier development in morphology and a higher density than that in the anal side. The density of ICC altered obviously during postnatal period, and the estimated total amount of ICC increased ~30 folds at P56 than that at P0. Some Kit⁺/Ki67⁺ and Kit⁺/BrdU⁺ cells were observed in ICC–MY, ICC–IM and ICC–SS, but not in ICC–SM from P0 to P24. Our result indicates a proximal to distal and transmural gradient development of ICC in the postnatal colon along with a dramatic increase of ICC cell number from neonatal to adult life, and an age-dependent proliferation of ICC is also involved.     

Development of the enteric nervous system,smooth muscle and interstitial cells of Cajal in the human gastrointestinal tract

The generation of functional neuromuscular activity within the pre-natal gastrointestinal tract requires the coordinated development of enteric neurons and glial cells, concentric layers of smooth muscle and interstitial cells of Cajal (ICC). We investigated the genesis of these different cell types in human embryonic and fetal gut material ranging from weeks 4–14. Neural crest cells (NCC), labelled with antibodies against the neurotrophin receptor p75^NTR, entered the foregut at week 4, and migrated rostrocaudally to reach the terminal hindgut by week 7. Initially, these cells were loosely distributed throughout the gut mesenchyme but later coalesced to form ganglia along a rostrocaudal gradient of maturation; the myenteric plexus developed primarily in the foregut, then in the midgut, and finally in the hindgut. The submucosal plexus formed approximately 2–3 weeks after the myenteric plexus, arising from cells that migrated centripetally through the circular muscle layer from the myenteric region. Smooth muscle differentiation, as evidenced by the expression of -smooth muscle actin, followed NCC colonization of the gut within a few weeks. Gut smooth muscle also matured in a rostrocaudal direction, with a large band of -smooth muscle actin being present in the oesophagus at week 8 and in the hindgut by week 11. Circular muscle developed prior to longitudinal muscle in the intestine and colon. ICC emerged from the developing gut mesenchyme at week 9 to surround and closely appose the myenteric ganglia by week 11. By week 14, the intestine was invested with neural cells, longitudinal, circular and muscularis mucosae muscle layers, and an ICC network, giving the fetal gut a mature appearance.A.S.W. is funded by a PhD studentship awarded to A.J.B. by the Child Health Research Appeal Trust.     

Relationships between neurokinin receptor-expressing interstitial cells of Cajal and tachykininergic nerves in the gut

The so-called interstitial cells of Cajal (ICC) are distributed throughout the muscle coat of the alimentary tract with characteristic intramural location and species-variations in structure and staining. Several ICC sub-types have been identified: ICC-DMP, ICC-MP, ICC-IM, ICC-SM. Gut motility is regulated by ICC and each sub-type is responsible for the electrical activities typical of each gut region and/or muscle layer. The interstitial position of the ICC between nerve endings and smooth muscle cells has been extensively considered. Some of these nerve endings contain tachykinins. Three distinct tachykinin receptors (NK1r, NK2r and NK3r) have been demonstrated by molecular biology. Each of them binds with different affinities to a series of tachykinins (SP, NKA and NKB). In the ileum, SP-immunoreactive (SP-IR) nerve fibers form a rich plexus at the deep muscular plexus (DMP), distributed around SP-negative cells, and ICC-DMP intensely express the SP-preferred receptor NK1r; conversely a faint NK1r-IR is detected on the ICC-MP and mainly after receptor internalization was induced by agonists. ICC-IM are never stained in laboratory mammals, while those of the human antrum are NK1r- IR. RT-PCR conducted on isolated ileal ICC-MP and gastric ICC-IM showed that these cells express NK1r and NK3r. Colonic ICC, except those in humans, do not express NK1r-IR, at least in resting conditions. Outside the gut, NK1r-IR cells were seen in the arterial wall and exocrine pancreas. In the mouse gut only, NK1r-IR is present in non-neuronal cells located within the intestinal villi, so-called myoid cells, which are c-kit-negative and alpha-smooth muscle actin-positive. Immunohistochemistry and functional studies confirmed that ICC receive input from SP-IR terminals, with differences between ICC sub-types. In the rat, very early after birth, NK1r is expressed by the ICC-DMP and SP by the related nerve varicosities. Studies on pathological conditions are few and those on mutant strains practically absent. It has only been reported that in the inflamed ileum of rats the NK1r-IR ICC-DMP disappear and that at the peak of inflammatory conditions ICC-MP are NK1r-IR. In the ileum of mice with a mutation in the W locus, ICC-DMP were seen to express c-kit-IR but not NK1-IR, and SP-IR innervation seems unchanged. In summary, there are distinct ICC populations, each of them under a different tachykininergic control and, likely, having different functions. Further studies are recommended at the aim of understanding ICC involvement in modulating/transmitting tachykininergic inputs.     

Structural characterization of interstitial cells of Cajal in myenteric plexus and muscle layers of canine colon

We have carried out a detailed ultrastructural study of the interstitial cells near the myenteric plexus of the canine colon and defined the structural characteristics which distinguish them from other resident non-neural cells. We have also examined the interconnections of these interstitial cells with nerves, the longitudinal muscle, and the circular muscle. In addition, we sought connections between interstitial cells of the myenteric plexus and those described earlier at the inner border of the circular muscle in proximal and distal colon. The interstitial cells of the myenteric plexus were structurally distinctive, and made gap junctions with one another and occasionally with smooth muscle. There seemed to be two subsets of these interstitial cells, one associated with the longitudinal muscle and the other with the circular muscle. Cells of both subsets were often close (less than or equal to 20 nm) to nerve profiles. The interstitial cells near the longitudinal muscle layer penetrated slightly into the muscle layer, but those near the circular muscle did not and neither set contacted the other. Moreover, interstitial cells of Cajal located near the myenteric plexus were never observed to contact those at the inner border of circular muscle. The interstitial cells of Cajal at the canine colon myenteric plexus are structurally organized to provide independent pacemaking activities for the longitudinal and adjacent circular muscle. Their dense innervation suggests that they mediate neural modulation of intestinal pacemaker activities. Moreover, they lack direct contacts with the interstitial cell network at the inner border of circular muscle, which is essential for the primary pacemaking activity of circular muscle. The structural organization of interstitial cells in canine colon is consistent with their proposed role in pacemaking activity of the two muscle layers.