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     

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.).     

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.     

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.     

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.     

Relationship between interstitial cells of Cajal and enteric motor neurons in the murine proximal colon

Interstitial cells of Cajal (ICC) are interposed between enteric neurons and smooth muscle cells in gastrointestinal (GI) muscles. The specific relationships between these cells in the murine proximal colon were studied with conventional and immunoelectron microscopy and immunohistochemistry. Intramuscular interstitial cells (IC-IM) formed discrete networks within the circular muscle layer of the murine proximal colon. Nerve trunks ran in close association with IC-IM and individual nerve trunks came into close contact with multiple IC-IM. Conventional electron microscopy revealed very close (< or = 20 nm) associations between nerve fibers and IC-IM. Processes of IC-IM also formed close contacts with neighboring smooth muscle cells. At the points of close association between neurons and IC-IM, areas of membrane densification in both pre- and postjunctional cells were present, suggesting specialized contacts or synaptic-like structures. Similar points of contact between neurons and smooth muscle cells were extremely rare. Immunoelectron microscopy demonstrated that IC-IM formed close associations with neurons containing nitric oxide synthase-like immunoreactivity (NOS-LI) or vesicular acetylcholine transporter-like immunoreactivity (vAChT-LI), suggesting innervation by both inhibitory and excitatory motor neurons. IC-IM were also labeled with anti-NOS antibodies. These observations suggest that IC-IM are an integral part of the neuromuscular junction in the colon. These cells may be the primary site of innervation, and neural regulation of the musculature may occur via IC-IM.     

CD34 immunoreactivity and interstitial cells of Cajal in the human and mouse gastrointestinal tract

Immunoreactivity for the tyrosine kinase receptor Kit (Kit-ir) is an established marker for the interstitial cells of Cajal (ICC) of the gut. Recently, the presence of CD34 immunoreactivity (CD34-ir) has been reported in Kit-ir ICC around the myenteric plexus in human small intestine. Conversely, we observed that CD34-ir labeled Kit-negative fibroblast-like cells, closely adjacent to, but distinct from, the Kit-ir ICC. The existence of cells expressing both CD34-ir and Kit-ir remains controversial. CD34-ir and Kit-ir were studied by high-resolution confocal microscopy on cryostat sections of human and murine gut as well as murine whole-mounts, using specific antibodies raised to human and murine CD34, respectively. CD34-ir labeled numerous cells in all parts of the gut, in man and in mouse. CD34-ir was consistently observed in Kit-negative cells, distinct from the closely adjacent Kit-ir ICC. Thin processes of both cell types intermingled extensively, often at the limit of resolution for light microscopy. CD34-ir was also observed in Kit-negative mesenchymal cells in the submucosa, in capillaries and in mesothelial cells. CD34-ir is not a marker for Kit-ir ICC in the human and murine gut. No CD34-ir, Kit-ir-expressing cells were encountered. Conversely, CD34-ir cells, closely adjacent to, but distinct from, Kit-ir ICC were consistently identified. The intimate relationship between these cells may offer an alternative explanation for reports of CD34 and Kit co-localization. The ontogeny and function of CD34-ir cells in the gut, as well as the origin of gastrointestinal stromal tumors, remain unclear.     

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

Electrical and mechanical activity of the circular muscle layer in the rectoanal region of the gastrointestinal tract undergoes considerable changes in the site of dominant pacemaking activity, frequency, and waveform shape. The present study was performed to determine whether changes in the structural organization of the circular layer or in the density, distribution, and ultrastructure of interstitial cells of Cajal (ICC) could account for this heterogeneity in electrical and mechanical activities. Light microscopy revealed that the structural organization of the circular muscle layer underwent dramatic morphological changes, from a tightly packed layer with poorly defined septa in the proximal rectum to one of discrete muscle bundles separated by large septae in the internal anal sphincter. Kit immunohistochemistry revealed a dense network of ICC along the submucosal and myenteric borders in the rectum, whereas in the internal anal sphincter, ICC were located along the periphery of muscle bundles within the circular layer. Changes in electrical activity within the circular muscle layer can be partially explained by changes in the structure of the muscle layer and changes in the distribution of ICC in the rectoanal region of the gastrointestinal tract.     

Neurotensin modulates pacemaker activity in interstitial cells of Cajal from the mouse small intestine

Neurotensin, a tridecapeptide localized in the gut to discrete enteroendocrine cells of the small bowel mucosa, is a hormone that plays an important role in gastrointestinal secretion, growth, and motility. Neurotensin has inhibitory and excitatory effects on peristaltic activity and produces contractile and relaxant responses in intestinal smooth muscle. Our objective in this study is to investigate the effects of neurotensin in small intestinal interstitial cells of Cajal (ICC) and elucidate the mechanism. To determine the electrophysiological effects of neurotensin on ICC, whole-cell patch clamp recordings were performed in cultured ICC from the small intestine. Exposure to neurotensin depolarized the membrane of pacemaker cells and produced tonic inward pacemaker currents. Only neurotensin receptor1 was identified when RT-PCR and immunocytochemistry were performed with mRNA isolated from small intestinal ICC and c-Kit positive cells. Neurotensin-induced tonic inward pacemaker currents were blocked by external Na⁺- free solution and in the presence of flufenamic acid, an inhibitor of non-selective cation channels. Furthermore, neurotensin-induced action is blocked either by treatment with {"type":"entrez-nucleotide","attrs":{"text":"U73122","term_id":"4098075","term_text":"U73122"}}U73122, a phospholipase C inhibitor, or thapsigargin, a Ca²⁺-ATPase inhibitor in ICC. We found that neurotensin increased spontaneous intracellular Ca²⁺ oscillations as seen with fluo4/AM recording. These results suggest that neurotensin modulates pacemaker currents via the activation of non-selective cation channels by intracellular Ca²⁺-release through neurotensin receptor1.     

Identification and classification of interstitial cells in the canine proximal colon by ultrastructure and immunocytochemistry

The ultrastructure and immunocytochemistry of interstitial cells (ICs) in the canine proximal colon were investigated. Three types of ICs were found within the tunica muscularis. (1) ICs were located along the submucosal surface of the circular muscle (IC-SM). These cells shared many features of smooth muscle cells, including myosin thick filaments and immunoreactivity to smooth muscle gamma actin, myosin light chain, and calponin antibodies. IC-SM were clearly different from smooth muscle cells in that contractile filaments were less abundant and intermediate filaments consisted of vimentin instead of desmin. (2) ICs in the region of the myenteric plexus (IC-MY) were similar to IC-SM, but these cells had no thick filaments or immunoreactivity to smooth muscle gamma actin or calponin antibodies. (3) The fine structures and immunoreactivity of ICs within the muscle layers (IC-BU) were similar to IC-MY, but IC-BU lacked a definite basal lamina and membrane caveolae. IC-BU and IC-MY were both immunopositive for vimentin. Since all ICs were immunopositive for vimentin, vimentin antibodies may be a useful tool for distinguishing between ICs and smooth muscle cells. Each class of ICs was closely associated with nerve fibers, made specialized contacts with smooth muscle cells, and formed multicellular networks. A combination of ultrastructural and immunocytochemical techniques helps the identification and classification of ICs by revealing the fine structures and determining the chemical coding of each class of ICs.     

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.     

Distribution and morphological characteristics of the interstitial cells of Cajal in the ileocaecal junction of the guinea-pig

The guinea-pig ileocaecal junction including the valve was studied by immunohistochemistry to clarify the organization of the muscle bundles, the enteric nerves and the interstitial cells of Cajal (ICC). This region clearly exhibited characteristic features in the distribution patterns of ICC in a proximal to distal direction: (1) the thickened portion of the terminal ileum immediately adjacent to the ileocecal junction contained many ICC throughout the circular (ICC-CM) and longitudinal (ICC-LM) muscle layers, but ICC were few or absent in the rest of the ileum; (2) the ileal side of the valve contained ICC associated with the deep muscular plexus (ICC-DMP) as in the small intestine, whereas ICC-DMP were absent in the caecal side as in the caecum; (3) the valve contained many ICC-CM and ICC-LM in both the ileal and caecal sides; (4) many ICC associated with the myenteric plexus were observed in both the ileal and caecal sides of the valve, whereas they were only sparsely found in the caecum; (5) ICC were also observed around the submucosal plexus in a confined area of the terminal ileum and the ileocaecal valve. These observations provide morphological evidence that the terminal ileum and ileocaecal valve are specially equipped for their active involvement in the movement of the junctional area.     

Carbachol regulates pacemaker activities in cultured interstitial cells of Cajal from the mouse small intestine

We studied the effect of carbachol on pacemaker currents in cultured interstitial cells of Cajal (ICC) from the mouse small intestine by muscarinic stimulation using a whole cell patch clamp technique and Ca²⁺-imaging. ICC generated periodic pacemaker potentials in the current-clamp mode and generated spontaneous inward pacemaker currents at a holding potential of–70 mV. Exposure to carbachol depolarized the membrane and produced tonic inward pacemaker currents with a decrease in the frequency and amplitude of the pacemaker currents. The effects of carbachol were blocked by 1-dimethyl-4-diphenylacetoxypiperidinium, a muscarinic M₃ receptor antagonist, but not by methotramine, a muscarinic M₂ receptor antagonist. Intracellular GDP-β-S suppressed the carbachol-induced effects. Carbachol-induced effects were blocked by external Na⁺-free solution and by flufenamic acid, a non-selective cation channel blocker, and in the presence of thapsigargin, a Ca²⁺-ATPase inhibitor in the endoplasmic reticulum. However, carbachol still produced tonic inward pacemaker currents with the removal of external Ca²⁺. In recording of intracellular Ca²⁺ concentrations using fluo 3-AM dye, carbachol increased intracellular Ca²⁺ concentrations with increasing of Ca²⁺ oscillations. These results suggest that carbachol modulates the pacemaker activity of ICC through the activation of non-selective cation channels via muscarinic M₃ receptors by a G-protein dependent intracellular Ca²⁺ release mechanism.     

Excitability of canine colon circular muscle disconnected from the network of interstitial cells of Cajal.

The 6 cpm omnipresent slow waves recorded in the circular muscle (CM) layer of canine colon are generated at the submucosal surface of the CM layer. After removal of the submucosal network of interstitial cells of Cajal (ICC), 66% of the CM preparations (25 of 38) were quiescent in Krebs solution. In the presence of carbachol, seven of nine of these spontaneously quiescent CM preparations demonstrated slow wave-like activity with mean frequency, duration and amplitude of 5.9 +/- 0.4 cpm, 2.8 +/- 0.5 s, and 0.8 +/- 0.2 mV, respectively. Similar slow wave-like activities were induced by TEA (seven out of eight quiescent CM preparations) with frequency, duration and amplitude of 6.1 +/- 0.2 cpm, 2.7 +/- 0.5 s, and 1.0 +/- 0.2 mV, respectively, and by BaCl2 (eight of eight quiescent CM preparations) with frequency, duration, and amplitude of 6.3 +/- 0.3 cpm, 1.8 +/- 0.2 s, and 0.5 +/- 0.1 mV, respectively. All the induced activities were abolished in the presence of 1 microM D600. CM preparations with the submucosal ICC network intact (ICC-CM) showed slow wave activity in Krebs solution at a frequency of 6.2 +/- 0.2 cpm, a duration of 3.6 +/- 0.2 s, and an amplitude of 1.0 +/- 0.1 mV (n = 22). When ICC-CM preparations were stimulated by BaCl2, carbachol, or TEA, the slow wave frequency did not change significantly, but the duration increased as well as the amplitude. In the presence of D600, the upstroke of slow waves remained and the frequency was not affected.(ABSTRACT TRUNCATED AT 250 WORDS)     

Volume-activated chloride currents in interstitial cells of Cajal

Interstitial cells of Cajal (ICC) undergo marked morphological changes on contraction of the musculature, making it essential to understand properties of mechanosensitive ion channels. The whole cell patch-clamp technique was used to identify and to characterize volume-activated Cl- currents in ICC cultured through the explant technique. Hypotonic solutions (approximately 210 mosM) activated an outwardly rectifying current, which reversed near the equilibrium potential for Cl-. Time-dependent inactivation occurred only at pulse potentials of +80 mV, with a time constant of 478 +/- 182 ms. The degree of outward rectification was calculated using a rectification index, the ratio between the slope conductances of +65 and -55 mV, which was 13.9 +/- 1.5 at 76 mM initial extracellular Cl- concentration. The sequence of relative anion permeability of the outwardly rectifying Cl- channel was I- > Cl- > aspartate-. The chloride channel blockers, DIDS and 5-nitro-2-(3-phenlypropl-amino)benzoic acid, caused a voltage-dependent block of the outwardly rectifying Cl- current, inhibition occurring primarily at depolarized potentials. On exposure to hypotonic solution, the slope conductance significantly increased at the resting membrane potential (-70 mV) from 1.2 +/- 0.2 to 2.0 +/- 0.4 nS and at the slow-wave plateau potential (-35 mV) from 2.1 +/- 0.3 to 5.0 +/- 1.0 nS. The current was constitutively active in ICC and contributed to the resting membrane potential and excitability at the slow-wave plateau. In conclusion, swelling or volume change will depolarize ICC through activation of outwardly rectifying chloride channels, thereby increasing cell excitability.     

Subunit b of cholera toxin labels interstitial cells of Cajal in the gut of rat and mouse

Cholera toxin subunit b was found in vivo and in vitro to label interstitial cells of Cajal in the intestine of rat and mouse. Cholera toxin-labelled interstitial cells were present in the subserosa, the myenteric plexus and the deep muscular plexus of mouse small intestine, and the deep muscular plexus only of the rat small intestine. In the large intestine of the mouse, interstitial cells were present in the subserosa and in a plexus associated with the inner surface of the circular muscle, while in the rat they were only present in the latter location. Macrophages, which were present in many of the same locations as interstitial cells, were also labelled by cholera toxin but could be distinguished from interstitial cells by their ability to take-up fluorescein isothiocyanate-labelled dextran. Labelling with subunit b of cholera toxin is a simple way of labelling interstitial cells of Cajal and which is compatible with a range of physiological and histological procedures.     

Innervation of interstitial cells of Cajal by vasoactive intestinal polypeptide containing nerves in canine colon.

The hypothesis was tested, through structural and functional studies, that interstitial cells of Cajal receive and can respond to direct innervation from nerves containing the vasoactive intestinal polypeptide neuromediator. The submucosal network of interstitial cells of Cajal has been postulated to provide pacemaking activity for the circular muscle and to be involved in neurotransmission from nonadrenergic, noncholinergic nerves for which vasoactive intestinal polypeptide is a putative mediator. The distribution of vasoactive intestinal polypeptide and substance P immunoreactive material in nerve profiles of the enteric nervous system of the canine colon was examined. In addition, electrophysiological studies were done on the interstitial cells bordering the submucosal side of the circular muscle layer after they were electrically isolated using heptanol. The vasoactive intestinal polypeptide immunoreactivity, located exclusively in nerve large granular vesicles, was found throughout the enteric nervous system (myenteric plexus, submucous plexus, and circular muscle--submucosa interface). The highest proportion (38% compared with 22-24%) of profiles of large granular vesicles with vasoactive intestinal polypeptide immunoreactivity was found in nerve profiles of the circular muscle--submucosa interface. In contrast, substance P immunoreactivity was found in nerve profiles of myenteric plexus (33% of large granular vesicles were positive) but not associated with submucosal interstitial cell nerve network. The vasoactive intestinal polypeptide hyperpolarized interstitial cells by 9 mV when electrically isolated by 1 mM heptanol and markedly reduced (about 50%) their input membrane resistance. We conclude that the distribution of vasoactive intestinal polypeptide immunoreactivity and its action are consistent with a postulated role of the interstitial cells as a major site of neurally mediated inhibition of colonic pacemaker activity.