Interstitial cells of Cajal in the deep muscular plexus mediate enteric motor neurotransmission in the mouse small intestine

Interstitial cells of Cajal (ICC) provide important regulatory functions in the motor activity of the gastrointestinal tract. In the small intestine, ICC in the myenteric region (ICC-MY), between the circular and longitudinal muscle layers, generate and propagate electrical slow waves. Another population of ICC lies in the plane of the deep muscular plexus (ICC-DMP), and these cells are closely associated with varicose nerve terminals of enteric motor neurons. Here we tested the hypothesis that ICC-DMP mediate excitatory and inhibitory neural inputs in the small bowel. ICC-DMP develop largely after birth. ICC-DMP, with receptor tyrosine kinase Kit-like immunoreactivity, appear first in the jejunum and then in the ileum. We performed electrophysiological experiments on mice immediately after birth (P0) or at 10 days post partum (P10) to determine whether neural responses follow development of ICC-DMP. At P0, slow-wave activity was present in the jejunum, but neural responses were poorly developed. By P10, after ICC-DMP developed, both cholinergic excitatory and nitrergic inhibitory neural responses were intact. Muscles of P0 mice were also put into organotypic cultures and treated with a neutralizing Kit antibody. Neural responses developed in culture within 3–6 days in control muscles, but blocking Kit caused loss of ICC and loss of cholinergic and nitrergic neural responses. Non-cholinergic excitatory responses remained after loss of ICC-DMP. Our observations are consistent with the idea that cholinergic and nitrergic motor neural inputs are mediated, to a large extent, via ICC-DMP. Thus, ICC-DMP appear to serve a function in the small intestine that is similar to the role of the intramuscular ICC in the stomach.     

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.     

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.     

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.     

Interstitial cells of Cajal: once negligible players, now blazing protagonists

Cajal in 1889 described a network of anastomosing interstitial cells in the gut muscle coat and hypothesized that they were accessory primitive neurons exerting a direct regulatory effect on smooth muscle contraction. Reticularists (among them Golgi) sustained that this net was not an assembly of individual cells but a true syncytium and the foremost dissidents, such as Kolliker and Dogiel, declared they were connective tissue cells. Keith, the discoverer of the sino-atrial node, suggested that these cells "constitute a pacemaker system of the intestinal muscle". In the period 1925-1960, there were papers still discussing the role and nature of the interstitial cells. The majority of these papers, however, reflect the fight between neuronists and reticularists. Around 1960, the reality of the neurons was established by ultrastructural evidence and interstitial cells degraded to fibroblasts or Schwann cells. By 1970, electron microscopists began to pay attention to these cells (from now named ICC). Among them, I myself concluded that ICC have smooth muscle features and might well be pacemaker cells. In this period, vital methylene-blue staining followed by electron microscopy firmly identified the ICC as myoid cells and the zinc iodide-osmic acid method, used to stain neurons, was also excellent for ICC and, when applied for electron microscopy, confirmed the identity of these cells. In the meantime different ICC populations were found in the gut muscle coat with region-specific location and region-specific features. By 1980, ICC, revealing themselves as myoid cells, a nature far more exciting than former ones, underwent to a booming interest and also physiologists began to study them. At present, it has been proved that one population, distributed throughout the entire gut, plays a pacemaker role; a second population, located intramuscularly in the stomach, is involved in neurotransmission, and a third population, specific of the small intestine, is part of the intestinal stretch receptor. By 1980 up to day, the differentiating steps of these cells were studied and factors implied in their maturation during foetal life and in the maintenance of their differentiated state in adulthood were identified. There has been also a rapidly evolving knowledge of specific molecules which are expressed on ICC, some of which useful for ICC identification under light- and electron microscope with a relative facility, some functionally implicated in neurotransmission and others in metabolic pathways strictly related to specific ICC behaviours. The more recent studies are considering the possibility of an ICC plasticity, transdifferentiation and apoptosis, especially in view of a direct implication of these cells in certain disorders of gut motility. Perspectives for future research are mainly concerning ICC alterations in gastrointestinal diseases.     

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.     

Interstitial cells of Cajal in the gastrointestinal musculature of W mutant mice

Interstitial cells of Cajal (ICC) are important regulatory cells generating electrical rhythmicity and transducing neural signals in the gastrointestinal musculature. ICC express the proto-oncogene c-kit, a receptor tyrosine kinase, and can be examined morphologically using the c-Kit antibody. The c-kit gene is allelic with the murine white-spotting locus W, and the c-kit mutation (W mutation) affects various aspects of hematopoietic cells, germ cells, melanocytes, mast cells, and ICC. Heterozygous W/W( v) mutant mice lack a specific type of ICC and have been used to reveal its function. To search for a new model that lacks a specific type of ICC, we examined homozygous W( v)/W( v) black-eyed-white mice that are viable with anemia. Results showed the principal patterns of ICC deficiency were the same between the W/W( v) and W( v)/W( v) mutants. In the stomach of both mice, intramuscular ICC (ICC-IM) were missing and myenteric ICC (ICC-MY) were reduced in number. In the small intestine, the number of ICC-MY was severely reduced in spite of a normal distribution of deep muscular plexus ICC (ICC-DMP). The cecum also exhibited fewer reduced. ICC-IM in the colon were almost entirely missing, whereas ICC-MY were reduced only in the distal colon. In the small intestine and colon, the number of remaining ICC-MY in W( v)/W( v) mice was greater than that in W/W( v) mice. The enteric nervous system of the two mutant mice showed normal characteristics. From these findings, we conclude that W( v)/W( v) mice represent a new genotype that lacks a part of the ICC in its gastrointestinal musculature.     

Interstitial cells of Cajal and electrical activity of smooth muscle in porcine ileum

Aim: To identify the interstitial cells of Cajal in the porcine ileum for the first time immunohistochemically and to examine the electrical properties of intestinal smooth muscle in the same region. Methods: In vitro intracellular microelectrode recordings were made from smooth muscle cells in cross‐sectional preparations from abattoir‐derived healthy porcine ileum. Immunohistochemical labelling of interstitial cells of Cajal was performed using an anti‐Kit antibody. Results: Slow waves were recorded in the circular muscle layer of all ileal preparations. The mean resting membrane potential of smooth muscle cells was ?61.0 ± 1.3 mV. Slow waves had a mean amplitude of 8.5 ± 0.5 mV, a frequency of 9.9 ± 0.1 cycles per minute and a duration of 5.6 ± 0.1 s. A waxing and waning pattern of slow wave activity was occasionally observed. In addition, higher frequency spiking activity associated with contractions was observed in some recordings. The L‐type calcium channel blocker nifedipine abolished both the spiking activity and the contractions, but had no significant effect on slow wave characteristics. Current‐injection manipulation of the resting membrane potential had no effect on slow wave amplitude, frequency or duration. Kit‐immunoreactive interstitial cells of Cajal were identified in the ileal samples and were present in the region of the myenteric plexus and in the circular and longitudinal muscle layers. Conclusion: This study recorded slow waves in vitro and demonstrated immunohistochemically the presence of interstitial cells of Cajal in the normal porcine ileum. This study forms a basis for future physiological and pathophysiological comparative studies of intestinal motility.     

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 in health and disease. Part II: ICC and gastrointestinal stromal tumours

Mesenchymal tumours in the gastrointestinal tract have long been problematic in terms of diagnosis, prognosis and therapy, but recent advances in immunohistochemistry and related therapies have allowed more specific diagnosis. In particular, the recognition that both the interstitial cells of Cajal (ICC) and many gastrointestinal stromal tumours (GISTs) are positive for c-kit and CD34 and have other features similar to those of ICC has led to the use of imatinib, a novel small molecule therapy that blocks the CD117/c-kit tyrosine kinase receptor, which shows remarkable efficacy in treatment of malignant and metastatic GISTs as well as other malignancies.     

Cajal间质细胞在胆囊结石形成中的作用分析

目的 观察Cajal间质细胞(interstitial cell of Cajal,ICC)和c-kit蛋白在正常胆囊和结石胆囊中的分布和表达情况,探讨ICC与胆囊结石发病的关系.方法 对手术切除的25例结石胆囊和15例非结石胆囊石蜡包埋组织标本和新鲜组织,通过c-kit免疫组化染色和Western blot,对两组胆囊标本ICC和c-kit蛋白进行检测.结果 所有胆囊标本中均发现c-kit阳性的ICC分布于胆囊平滑肌层,结石胆囊ICC数目和c-kit蛋白表达明显少于非结石胆囊.结论 胆囊结石发病过程中胆囊动力学的减弱有可能与ICC的减少有一定关联.     

Interstitial cells of Cajal reduce in number in recto-sigmoid Hirschsprung's disease and total colonic aganglionosis

Interstitial cells of Cajal (ICCs) play a key role in regulating gastrointestinal tract motility. The pathophysiological basis of colonic aperistalsis in Hirschsprung's disease (HD) is still not fully understood. Many studies reported that decreased numbers or disrupted networks of ICCs were associated with HD. Little information is available on the distribution of different subtypes of ICCs in HD. The aim of this study was to determine the alterations in density of different subtypes of ICC in colonic specimens of patients with total colonic and recto-sigmoid HD. Full thickness colonic specimens were obtained from five children with total colonic aganglionosis (TCA), sixteen with recto-sigmoid HD and seven controls. ICCs were visualized in frozen sections by c-Kit (CD117) fluorescent staining. In the control colon, c-Kit positive ICCs formed a dense network surrounding the myenteric plexus (IC-MY), along the submucosal surface of the circular muscle layer (IC-SM) and in the circular and longitudinal muscle layer (IC-IM). In the aganglionic region of the colon of the patients affected by HD, the number of ICCs (especially IC-IM and IC-SM) was markedly reduced and IC-MY networks were disrupted. Nearly total lack of three subtypes of ICCs was observed in the TCA specimens. This study demonstrated the altered distribution of different subtypes of ICCs in the resected colon of patients with recto-sigmoid HD and TCA. These findings suggest that the reduction of each subtype of ICCs may play an important role in the etiology of HD.     

The involvement of the interstitial Cajal cells and the enteric nervous system in bowel endometriosis

BACKGROUND: Our aim was to investigate the relationships between gastrointestinal symptoms and histological findings in women with bowel endometriosis. METHODS: The gastrointestinal symptoms of 362 women with endometriosis were classified according to the subgroups of the Rome II criteria. All visible endometriotic lesions of the bowel were removed; the patients were prospectively followed up for 2 years. The interstitial Cajal cells (ICC) and the enteric nervous system were immunohistochemically evaluated. RESULTS: Sixty-eight (18.8%, 95% CI 14.9-23.2) women had bowel lesions. The endometriotic lesions infiltrated the serosal layer and surrounding connective tissue in 45 cases; the subserous plexus in 11 cases; the Auerbach plexus in eight cases; the Meissner plexus in four cases. Whenever the subserous plexus was interrupted by the endometriotic lesions, the ICC were damaged. All women with endometriotic lesions reaching at least the subserous plexus reported bowel complaints. The level of infiltration into the bowel wall was correlated with severity of symptoms. Removal of lesions resulted in improvement of symptoms. CONCLUSIONS: Endometriosis-induced damage of ICC, even before muscular infiltration, may cause bowel symptoms.     

Nitrergic signalling via interstitial cells of Cajal regulates motor activity in murine colon

Key points

• Dysregulation of nitric oxide (NO) signalling is associated with GI motility dysfunctions like chronic constipation, achalasia or Hirschsprung''s disease. The inhibitory effect of NO is mainly exerted via NO‐sensitive guanylyl cyclase (NO‐GC) which is found in different gastrointestinal (GI) cell types including smooth muscle cells (SMCs) and interstitial cells of Cajal (ICC).
• Here, we focus on the investigation of NO‐GC function in murine colon. Using cell‐specific knock‐out mice, we demonstrate that NO‐GC is expressed in myenteric ICC of murine colon and participates in regulation of colonic spontaneous contractions in longitudinal smooth muscle.
• We report a novel finding that basal enteric NO release acts via myenteric ICC to influence the generation of spontaneous contractions whereas the effects of elevated endogenous NO are mediated by SMCS in the murine proximal colon.
• These results help in understanding possible pathological mechanisms involved in slowed colonic action and colonic inertia.

Abstract

In the enteric nervous systems, NO is released from nitrergic neurons as a major inhibitory neurotransmitter. NO acts via NO‐sensitive guanylyl cyclase (NO‐GC), which is found in different gastrointestinal (GI) cell types including smooth muscle cells (SMCs) and interstitial cells of Cajal (ICC). The precise mechanism of nitrergic signalling through these two cell types to regulate colonic spontaneous contractions is not fully understood yet. In the present study we investigated the impact of endogenous and exogenous NO on colonic contractile motor activity using mice lacking nitric oxide‐sensitive guanylyl cyclase (NO‐GC) globally and specifically in SMCs and ICC. Longitudinal smooth muscle of proximal colon from wild‐type (WT) and knockout (KO) mouse strains exhibited spontaneous contractile activity ex vivo. WT and smooth muscle‐specific guanylyl cyclase knockout (SMC‐GCKO) colon showed an arrhythmic contractile activity with varying amplitudes and frequencies. In contrast, colon from global and ICC‐specific guanylyl cyclase knockout (ICC‐GCKO) animals showed a regular contractile rhythm with constant duration and amplitude of the rhythmic contractions. Nerve blockade (tetrodotoxin) or specific blockade of NO signalling (l‐NAME, ODQ) did not significantly affect contractions of GCKO and ICC‐GCKO colon whereas the arrhythmic contractile patterns of WT and SMC‐GCKO colon were transformed into uniform motor patterns. In contrast, the response to electric field‐stimulated neuronal NO release was similar in SMC‐GCKO and global GCKO. In conclusion, our results indicate that basal enteric NO release acts via myenteric ICC to influence the generation of spontaneous contractions whereas the effects of elevated endogenous NO are mediated by SMCs in the murine proximal colon.

Abbreviations

DEA‐NO

2‐(N,N‐diethylamino)‐diazenolate‐2‐oxide diethylammonium salt

EFS

electrical field stimulation

GCKO

guanylyl cyclase knockout

ICC

interstitial cells of Cajal

ICC‐GCKO

ICC‐specific guanylyl cyclase knockout

KO

knockout

NO

nitric oxide

NO‐GC

nitric oxide‐sensitive guanylyl cyclase

l‐NAME

N ^G‐nitro‐l‐arginine methyl ester

nNOS

neuronal nitric oxide synthase

ODQ

1H‐[1,2,4]oxadiazolo[4,3‐a]quinoxalin‐1‐one

PKG

cGMP‐dependent protein kinase

SMC

smooth muscle cell

SMC‐GCKO

smooth muscle‐specific guanylyl cyclase knockout

TTX

tetrodotoxin

WT

wild‐type

     

Interstitial cells of Cajal in patients with chagasic megacolon originating from a region of old endemicity.

Megacolon is the second most frequent and most important digestive manifestation of Chagas' disease. It is characterized by motor disorders and dilatation of the distal segments of the colon. Several theories have been presented to explain the physiopathology of chagasic megacolon, e.g. the plexus theory. Objective: In the present study the distribution of interstitial cells of Cajal (ICC) was evaluated in colon biopsies from chronic chagasic patients originating from a region of old endemicity for Trypanosoma cruzi and for comparison in subjects with other colon disorders. The chagasic patients had been submitted to colectomy for the investigation of other possible mechanisms underlying the physiopathogenesis of megacolons. Design study: Twenty-two colon biopsies (15 from chagasic patients and 7 from nonchagasic patients) were examined. ICC were identified by immunohistochemistry by using the anti-CD117 antibody. The number of ICC was determined in longitudinal and circular muscle layers and in the myenteric plexus, and the results were analyzed by the Kruskal-Wallis and Student t-tests. Results: A reduced number of ICC was observed in all layers and in the myenteric plexus of patients with chagasic megacolon (P<0.05). Conclusions: We conclude that the physiopathological manifestations observed in the large bowel of chagasic patients originate from alterations that occur in the ICC, which play an important role in the control of gut motility.     

消化道蠕动的起搏器与Cajal间质细胞

消化道蠕动是消化道平滑肌群的节律性收缩活动。越来越多的研究证据表明,这种活动是由一类叫做(pacem aker)起搏器的特殊细胞引起的。这些细胞可能就是分布在消化道平滑肌层间的(interstitialcells of Cajal)ICC。它们因分布的部位不同而形态各异。但它们都含有丰富的线粒体,呈c-K it免疫染色阳性,并伸出很多突起形成网络。他们相互之间及与周围的平滑肌细胞之间以缝隙结合相连接。这篇综述主要介绍此类细胞的形态结构及分布特点。