Interstitial cells of Cajal: primary targets of enteric motor innervation

For many years morphologists have noted the close relationship between interstitial cells of Cajal (ICC) and nerve fibers within the tunica muscularis of gastrointestinal (GI) organs. These observations led to speculations about a role for ICC in mediating neural inputs to the GI tract. Immunohistochemical and functional studies demonstrated the presence of receptors for the neurotransmitters utilized by enteric motor neurons, and changes in second messengers in ICC after field stimulation of intrinsic enteric neurons showed that ICC were functionally innervated in GI muscles. Recent double labeling experiments have shown that both excitatory and inhibitory enteric motor neurons are closely associated with ICC in the deep muscular plexus (IC-DMP) of the small intestine and intramuscular ICC (IC-IM) of the proximal and distal GI tract. Enteric motor neurons form synaptic-like structures with IC-IM and IC-DMP. Far fewer close contacts are found between enteric motor neurons and smooth muscle cells. Experiments on W/W(V) mutants that lack IC-IM in the stomach, lower esophageal sphincter, and pylorus have shown that these ICC are critical components of the neuromuscular junction. Cholinergic excitatory and nitrergic inhibitory neurotransmission are severely decreased in tissues lacking IC-IM, yet there is no loss of cholinergic or nitrergic neurons in W/W(V) mutants. These data suggest that either the post-junctional mechanisms responsible for receiving and transducing neurotransmitter signals are specifically expressed by ICC, or that the large extracellular spaces typically between nerve terminals and smooth muscle cells may not allow effective concentrations of neurotransmitters to reach receptors expressed by smooth muscle cells. These findings indicate an important role for certain classes of ICC in enteric neurotransmission and predict that loss of ICC in human motor disturbances may significantly compromise neural regulation of GI motility.     

Further observations on the gap-junction-rich cells in the deep muscular plexus of the rat small intestine

 Interstitial cells forming many large gap junctions in the region of the deep muscular plexus of the rat small intestine were studied by electron microscopy and by three-dimensional cell models reconstructed from serial ultrathin sections. Two different profiles of cells were observed. Cells of the first profile are characterized by an elongated cell shape and by less electron-dense cytoplasm, containing many mitochondria, well-developed Golgi apparatus and free ribosomes. They mainly connect with smooth muscle cells of the main circular layer. In a three-dimensional cell model, the total area of the gap junctions occupies 1.3% of the cell surface. Cells of the second profile are characterized by the frequent occurrence of slender cytoplasmic processes, higher electron-dense cytoplasm, containing mitochondria, Golgie apparatus and well-developed rough endoplasmic reticulum, and numerous caveolae on the cell membrane. In this cell model, gap junctions occupy 0.8% of the cell surface. The ratio of gap junctions with the same profile of cells to the total gap junction area is 37.7%, which is more than three times greater than the 9.9% in cells of the first profile. These cells were closely associated with nerve terminals. It is likely that these cells with different profiles constitute subtypes with each other and cooperate for regulation of intestinal motility via the transmission of nerve signals. Accepted: 28 August 1997     

Interstitial cells of Cajal and Auerbach's plexus. A scanning electron microscopical study of guinea-pig small intestine.

Interstitial cells of Cajal (ICC) appear to be involved in the regulation of intestinal motility, probably as pacemaker cells. We investigated the complex organization of ICC associated with Auerbach's plexus of guinea-pig small intestine in the scanning electron microscope. The plexus was exposed by microdissection of zinc iodide/osmic acid stained tissue. After separation of the muscle layers by microdissection alone, the exposed Auerbach's plexus was seen to be covered by a smooth mat of reticular fibrils, thin enough to allow the detailed examination of the intact nerve plexus and interstitial tissue. ICC were distinguished as small, ovoid cell bodies from which 2-5 long, branching, roughly cylindrical processes emerge, associating to form a complex network. Characteristically, ICC processes participated in the formation of small bundles along their course, individual processes passing from one bundle to another. Cell bodies and processes of ICC were intimately associated with tertiary nerves of Auerbach's plexus. Axons were identified and distinguished these from ICC processes by their varicose structure and by th smaller diameters as compared with ICC processes. We found no single axons in our material. The characteristic morphology of ICC clearly distinguished these as a separate cell population different from neurons, glial cells, fibroblasts, and smooth muscle cells. After removal of the mat of reticular fibrils by chemical digestion the detailed organization of the interstitial tissue was preserved. Macrophage-like cells were previously demonstrated by other techniques to constitute a constant and rather dense population of cells in the studied location. As an indication that we preserve the full complement of interstitial cells by our technique, these macrophage-like cells wer for the first time identified in material processed for scanning electron microscopy. The cells had characteristically irregular cell surfaces with short, veil-like, folded extensions intertwined between the other cells in the interstices. Our study establishes an improved correlation between results obtained by the application of scanning electron microscopy to dissected tissue and results from light and transmission electron microscopy of the intact tissue.     

W/W c-Kit mutant mice possess interstitial cells of Cajal in the deep muscular plexus layer of the small intestine

The c-Kit receptor tyrosine kinase regulates the development and differentiation of various progenitor cells. W mutant mice with spontaneous mutations in the c-kit gene show various phenotypes such as anemia, infertility, loss of coat color and mast cells. c-Kit also regulates the development of the interstitial cells of Cajal (ICC) that are responsible for the motility regulation of the gastrointestinal musculature. W^sh/W^sh mice possess an inversion mutation upstream of the c-kit promoter region; this mutation is responsible for reducing c-Kit activity, leading to a decrease in the number of mast cells, melanocytes, and ICC. We extensively examined the small intestine of W^sh/W^sh mice by using immunohistochemistry and electron microscopy. Although the musculature of the W^sh/W^sh mice did not show any c-Kit immunoreactivity, there were neurokinin 1 receptor (NK1R)-immunopositive cells that were associated with the nerve fibers in the deep muscular plexus (DMP) region. These NK1R-immunopositive cells showed a bipolar shape with long processes and were identified as ICC in the DMP layer (ICC-DMP). Electron microscopic analysis revealed that ICC-DMP had numerous mitochondria, caveolae, and gap junctions and were closely associated with nerve terminals. In contrast, ICC were not observed at the myenteric layer. In the small intestine of the W^sh/W^sh mice, we detected ICC-DMP that showed NK1R immunoreactivity and ultrastructural characters. This type of ICC may develop and maturate structurally without c-Kit expression and regulate gastrointestinal motility.     

Interstitial cells of Cajal are functionally innervated by excitatory motor neurones in the murine intestine

Recent studies have demonstrated that intramuscular interstitial cells of Cajal (ICC) are preferential targets for neurotransmission in the stomach. Terminals of enteric motor neurones also form tight, synaptic-like contacts with ICC in the small intestine and colon, but little is known about the role of these cells in neurotransmission. ICC at the deep muscular plexus (ICC-DMP) of the small intestine express neurokinin 1 receptors (NK1R) and internalize these receptors in response to exogenous substance P. We used NK1R internalization as an assay of functional innervation of ICC-DMP in the murine small intestine. Under basal conditions NK1R-like immunoreactivity (NK1R-LI) was mainly observed in ICC-DMP (519 cells counted, 100% were positive) and myenteric neurones. ICC-DMP were closely apposed to substance P-containing nerve fibres. Of 338 ICC-DMP examined, 65% were closely associated with at least one substance P-positive nerve fibre, 32% were associated with at least two, 2% were associated with more than two nerve fibres and 1% with none. After electrical field stimulation (EFS, 10 Hz; 1 min) NK1R-LI was internalized in more than 80% of ICC-DMP, as compared to 10% of cells before EFS. Internalization of NK1R was not observed in myenteric ICC or smooth muscle cells in response to nerve stimulation. Internalization of NK1R-LI was blocked by the specific NK1 receptor antagonist WIN 62577 (1 μ m ) and by tetrodotoxin (0.3 μ m ), suggesting that internalization resulted from stimulation of receptors with neurally released neurokinins. These data suggest that ICC-DMP are primary targets for neurokinins released from enteric motor neurones in the intestine.     

The cellular network of interstitial cells associated with the deep muscular plexus of the guinea pig small intestine

Summary Systematic examination using electron microscopic montages and serial sections has demonstrated that three types of interstitial cell, namely gap junction-rich cells, glycogen-rich cells and fibroblast-like cells, are densely located along the whole extent of the deep muscular plexus of the guinea pig small intestine. They tend to be distributed in an alternating fashion in the cellular network, connected with muscle cells of the outer, circular layer by means of gap junctions. These three types of interstitial cell show close relations to two types of nerve varicosity: one type is characterized by clear round vesicles with diameters of about 50 nm, and the other by flattened vesicles measuring about 35 nm by 70 nm. Electron-dense patches have been observed at the cytoplasmic side of the axonal membranes. Muscle cells of both inner and outer circular layers also show close relations to these two types of nerve varicosity. These morphological features are discussed with the implication that they may have some regulatory role in intestinal movement.     

Interstitial cells of Cajal associated with the submucosal plexus of the Guinea-pig stomach

Interstitial cells of Cajal (ICC) form specialized networks in the gastrointestinal tract that coordinate cellular communications between nerves and smooth muscle cells. However, little is known about ICC in the gut mucosa or submucosa. Here, we report for the first time that Kit-immunoreactive ICC are associated with the submucosal (Meissner's) plexus of the Guinea-pig stomach. In longitudinal sections along the greater curvature of the gastric corpus, short spindle-shaped ICC of the submucosal plexus (ICC-SP) were located around the PGP9.5-immunoreactive nerve elements in the submucosa. Observations of whole-mount preparations clearly demonstrated Kit-immunoreactive bipolar or multipolar cells with long cytoplasmic processes about 100 microm in length. Such cells had typical characteristics of ICC, confirming that they were not mast cells, which are also Kit-immunoreactive residents of the submucosal connective tissue space. Although some ICC-SP surrounded parts of the submucosal plexus, they did not appear to form wide extensions of the cellular network, suggesting that they acted locally. The demonstration of ICC-SP in the submucosal connective tissue space suggests that they may contribute to the regulation of secretion, absorption and transportation of fluids in the mucosa.     

Interstitial cells of Cajal, enteric nerves, and glial cells in colonic diverticular disease

BACKGROUND: Colonic diverticular disease (diverticulosis) is a common disorder in Western countries. Although its pathogenesis is probably multifactorial, motor abnormalities of the large bowel are thought to play an important role. However, little is known about the basic mechanism that may underlie abnormal colon motility in diverticulosis. AIMS: To investigate the interstitial cells of Cajal (the gut pacemaker cells), together with myenteric and submucosal ganglion and glial cells, in patients with diverticulosis. PATIENTS: Full thickness colonic samples were obtained from 39 patients undergoing surgery for diverticulosis. Specimens from tumour free areas of the colon in 10 age matched subjects undergoing surgery for colorectal cancer served as controls. METHODS: Interstitial cells of Cajal were assessed using anti-Kit antibodies; submucosal and myenteric plexus neurones and glial cells were assessed by means of anti-PGP 9.5 and anti-S-100 monoclonal antibodies, respectively. RESULTS: Patients with diverticulosis had normal numbers of myenteric and submucosal plexus neurones compared with controls (p = 0.103 and p = 0.516, respectively). All subtypes of interstitial cells of Cajal were significantly (p = 0.0003) reduced compared with controls, as were glial cells (p = 0.0041). CONCLUSIONS: Interstitial cells of Cajal and glial cells are decreased in colonic diverticular disease, whereas enteric neurones appear to be normally represented. This finding might explain some of the large bowel motor abnormalities reported to occur in this condition.     

Classes of enteric nerve cells in the guinea-pig small intestine

The guinea-pig small intestine has been very widely used to study the physiology, pharmacology and morphology of the enteric nervous system. It also provides an ideal, simple mammalian preparation for studying how nerve cells are organised into functional circuits underlying simple behaviours. Many different types of nerve cells are present in the enteric nervous system and they show characteristic combinations of morphological features, projections, biophysical properties, neurochemicals, and receptors. To identify the different functional classes is an important prerequisite for systematic analysis of how the enteric nervous system controls normal gut behaviour. Based on combinations of multiple-labelling immunohistochemistry and retrograde tracing, it has been possible to account quantitatively for all of the neurones in the guinea-pig small intestine. This article summarises that account and updates it in the light of recent data. A total of 18 classes of neurones are currently distinguishable, including primary afferent neurones, motor neurones, interneurones, secretomotor and vasomotor neurones. It is now possible to take an individual nerve cell and use a few carefully chosen criteria to assign it to a functional class. This provides a firm anatomical foundation for the systematic analysis of how the enteric nervous system normally functions and how it goes wrong in various clinically important disorders.     

Interstitial cells of Cajal in chagasic megaesophagus

Chagasic visceromegalies are the most important digestive manifestations of Chagas disease and are characterized by motor disorders and dilation of organs such as esophagus and colon. One of the theories raised to explain the physiopathogenesis of chagasic megas is the plexus theory. Recent studies have shown a reduction of interstitial cells of Cajal (ICCs) in the colon of chagasic patients. These cells are present throughout the gastrointestinal tract and are considered to be pacemaker cells, that is, they are responsible for coordinating peristalsis and for mediating nerve impulses. In view of the lack of studies on these cells in megaesophagus and the previous observation of a reduction of ICCs in chagasic megacolons, we compared the distribution of ICCs in the esophagus of chagasic and nonchagasic patients to contribute to a better understanding of the physiopathogenesis of this esophageal disease. Esophageal biopsy samples from 10 chagasic and 5 nonchagasic patients were used. Cells were identified with the anti-CD117 antibody. The number of ICCs was quantified in longitudinal and circular muscle layers and myenteric plexus. The results were analyzed statistically by comparison of means. An intense reduction in the number of ICCs was observed in muscle layers and in the myenteric plexus of patients with megaesophagus. We conclude that there is an intense reduction of ICCs in the esophagus of chagasic patients when compared to nonchagasic patients, a finding supporting the important role of these cells in gastrointestinal tract motility. A deficiency in these cells might be implied in the genesis of megaesophagus.     

Intestitial cells of Cajal in the human small intestine: immunochemical and ultrastructural study

The stem cell kinase CD117 has recently been found to play an important role in the development of interstitial cells of Cajal (ICC), which are currently regarded as pacemaker cells of the gastrointestinal tract. CD117 is expressed in both gastrointestinal stromal tumors (GIST) and ICC, with the latter regarded by many as the progenitor cells of GIST. The authors investigated immunoreactivity of 25 normal surgically removed small intestinal tissues and correlated the findings with electron microscopy (EM) on 12 cases. In all cases CD117-positive cells were frequently seen around the myenteric plexi either singly or in groups. CD117-positive cells on immunostained sections corresponded to the cells appearing as fibroblast-like or undifferentiated primitive mesenchymal cells around the myenteric ganglia and interstitial spaces by EM. In contrast, S-100 stain revealed a fine network of positive staining throughout the muscularis. Branches of nonmyelinated axons and nerve endings were found regularly between myocytes with direct contact with muscle cells by EM. The cells that we could depict as ICC because of their distribution andstaining pattern of CD117 were limited to the nonmuscular mesenchymal cells. No muscle cell-like ICC were found. Instead, the muscle cells in direct contact with nerve endings were often disfigured and the cytoarchitectural contents for muscle cells became less distinct because of lighter staining and loss of definite focal densities among actin filaments. However, these latter cells did maintain most muscle cell features, such as continuous external lamina, caveolae, and some of the peripheral densities. These findings raise a possibility that previous investigators could have included these altered muscle cells into the ICC group. It was also found that intestinal muscularis not only was richly endowed with an elaborate neural network of delicate axonal extensions and dense-core granule containing nerve endings traversing through and between myocytes, but also showed frequent synapse-like direct contact between nerve endings and muscle cells. These findings indicate that enteric nerves may play a major role in the control of intestinal motility, while CD117-positive cells play an accessory role as cells of Cajal as originally speculated. Further studies are necessary to better define and characterize interstitial cells of Cajal, which will be useful in the correlation of the vast number of data concerning the possible role of CD117-positive ICC in the pacemaker function of the intestine and oncogenesis of GIST.     

Changes in interstitial cells of Cajal at the deep muscular plexus are associated with loss of distention-induced burst-type muscle activity in mice infected by Trichinella spiralis

The physiology and pathophysiology of the network of interstitial cells of Cajal associated with the deep muscular plexus (ICC-DMP) of the small intestine are still poorly understood. The objectives of the present study were to evaluate the effects of inflammation associated with Trichinella spiralis infection on the ICC-DMP and to correlate loss of function with structural changes in these cells and associated structures. We used immunohistochemistry, electron microscopy, and assessment of distention-inducing electrophysiological parameters in vitro. Damage to ICC-DMP was associated with a loss of distention-induced patterns of electrical activity normally associated with distention-induced peristalsis. Consistently, the timing of recovery of ICC-DMP paralleled the timing of recovery of the distention-induced activity. Nerve varicosities associated with ICC-DMP including cholinergic nerves, assessed by immunoelectron microscopy and whole mount double labeling, paralleled injury to ICC-DMP thus contributing to impaired excitatory innervation of smooth muscle cells. Major additional changes included a remodeling of the inner circular muscle layer, which may affect long-term sensitivity to distention after infection. In conclusion, transient injury to ICC-DMP in response to T. spiralis infection is severe and associated with a complete lack of distention-induced burst-type muscle activity.     

Macrophage-like cells in the muscularis externa of mouse small intestine

In muscularis externa of mouse small intestine, cells with ultrastructural features of macrophages were invariably observed in three layers: in the subserosal layer, between the circular and longitudinal muscle layers, and in association with the deep circular plexus. These macrophage-like cells (MLC) had a single indented nucleus, perinuclear Golgi complex, smooth and rough endoplasmic reticulum, many pits (coated and uncoated) in the plasma membrane, coated vesicles, light vesicles, and primary lysosomes, but rather few heterogeneous lysosomal vacuoles. MLC were partially enveloped by processes of interstitial cells of Cajal. FITC-dextran used in combined fluorescence stereo microscopy, fluorescence microscopy, and electron microscopy was employed as a tracer to study the endocytic qualities of the MLC. The mice were killed 5, 15, 30, and 60 min, 1 day, and 4 days after dextran administration. By fluorescence microscopy after 1 or 4 days MLC were observed as a constant cellular population with a strikingly regular distribution. By electron microscopy dextran-containing vacuoles were conspicuous after 1 h or more. MLC of the subserosal layer and between the circular and longitudinal muscle layers could be distinguished with respect to general appearance, pattern formation, and apparent dextran contents.     

Renewal of Paneth cells in the small intestine of the mouse

The origin and fate of Paneth cells were examined in duodenum, jejunum and ileum of adult female mice, using radioautography after administration of ³H-thymidine either in a single injection or in drinking water for four days or as a continuous infusion for up to ten days. The tissues were fixed by perfusion with 4% paraformaldehyde. One-micron thick, Eponembedded single or serial sections were stained with Regaud's hematoxylin, radioautographed, and counterstained with safranin O. Mitosis of Paneth cells is never observed, nor are these cells ever labeled one hour after ³H-thymidine. Hence, Paneth cells do not divide. However, a few days after single injection or prolonged administration of ³H-thymidine, labeled Paneth cells appear. The first labeled cells have tiny granules but, as the cells age, larger and larger granules are observed. Adjacent to Paneth cells are slender undifferentiated cells which show frequent mitoses and early labeling. The evidence points to some of these cells transforming into Paneth cells. Since occasionally Paneth cells degenerate, the newly-formed ones would provide replacement for those which die, thus insuring the steady state of the Paneth cell population. The renewal of this population is characterized by a turnover time of about three weeks.     

Interstitial cells of Cajal at the clinical and scientific interface

Considerable work over the past two decades has determined that interstitial cells of Cajal (ICC) serve as pacemaker cells, conduits for active transmission of electrical slow waves, sites of innervation by peripheral motor neurons, and mechanotransducers. While most of the physiology of ICC has been learned from studies of the cells within the gastrointestinal tract, ICC are found in a variety of smooth muscle tissues and may have analogous or novel physiological functions in those organs. Clinical investigations of muscles from patients with a variety of gastrointestinal motility disorders have raised the exciting possibility that loss of ICC may be responsible for the development of motor dysfunction. This review discusses the development of ICC, the kinds of human disorders in which ICC loss may be important, what factors regulate the ICC phenotype, and what therapeutic approaches might be utilized to restore or regenerate ICC. This field is primed for translational discoveries. ICC are responsible for critical physiological functions in smooth muscle tissues, they are lost in pathophysiological conditions, and it will be important now to decipher the conditions that are responsible for ICC loss and develop new therapies to relieve patients of this problem. Success in this endeavour might improve the quality of life for millions of patients.     

新生小鼠小肠Cajal间质细胞增殖的实验研究

目的 探讨在新生小鼠小肠Cajal间质细胞(interstitial cells of Cajal,ICC)的发育过程中是否出现增殖.方法 采用新生2 d(P2d)、14 d(P14d)和24 d(P24d)的小鼠小肠,应用BrdU腹腔注射,24 h后取材,Kit和BrdU免疫荧光染色.结果 P2d小鼠小肠,可见大量Kit/BrdU双重标记阳性细胞,这些细胞的形态与成熟ICC基本相似,这些细胞在P14d时减少,而在P24d消失.统计学分析发现,不同时间点之间Kit/BrdU双标阳性细胞数量存在显著差别(P＜0.05).结论 在小肠生后发育过程中,出现了ICC增殖,而这种增殖在发育过程中逐渐减弱,最后在成年前消失.     

Cytodifferentiation of the interstitial cells of Cajal related to the myenteric plexus of mouse intestinal muscle coat

Summary The cytodifferentiation of the interstitial cells of Cajal (Type I) related to the myenteric plexus of the mouse intestinal muscle coat was studied in foetuses at term, neonates not yet fed, suckling animals, weaning animals and adult animals. In foetuses at term, interstitial cells of Cajal and their precursor cells are not identifiable. In neonates not yet fed, presumed precursor cells of the interstitial cells of Cajal, i.e. ICC-blasts, can be identified as cells surrounding the developing myenteric plexus and interposed between the circular and longitudinal muscle layers. These ICC-blasts are poorly differentiated but, like the adult interstitial cells, are always related to nerve endings and possess large and numerous mitochondria. In suckling animals these cells gradually develop, and are fully differentiated only after the end of the weaning period.