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Interstitial cells of Cajal in the guinea-pig gastrointestinal tract as revealed by c-Kit
Abstract
Interstitial cells of Cajal (ICC) of various morphologies have been described in the gastrointestinal (GI) tracts of mammals. Different classes of ICC are likely to have different functional roles. ICC of the mouse GI tract have been shown to express c-kit, a proto-oncogene that codes for a receptor tyrosine kinase. We have studied the distribution of ICC within the guinea pig GI tract using antibodies to c-Kit protein and immunohistochemical techniques. c-Kit-like immunoreactivity revealed at least 6 types of ICC: (1) intramuscular ICC (IC-IM1) that lie within the muscle layers of the esophagus, stomach, and cecum, (2) ICC within the myenteric plexus region (IC-MY1) in the corpus, antrum, small intestine, and colon,(3) ICC that populate the deep muscular plexus of the small intestine (IC-DMP), (4) ICC at the submucosal surface of the circular muscle layer in the colon (IC-SM), (5) stellate ICC that are closely associated with the myenteric plexus (IC-MY2) and orientated toward the longitudinal muscle layer in the colon, and (6) branching intramuscular ICC (IC-IM2) in the proximal colon within the circular and longitudinal muscle layers. c-Kit immunohistochemistry appears to be an excellent and selective technique for labeling ICC of the guinea-pig GI tract. Labeling of these cells at the light-microscopic level provides an opportunity for characterizing the distribution, density, organization, and relationship between ICC and other cell types.
... Networks of interstitial cells of Cajal (ICC) were first described by Spanish neurologist Santiago Ramón y Cajal (Thuneberg 1999). They are considered to be a specialized population of smooth muscle cells embedded in and around the musculature of the GI tract (Burns et al. 1997;Maeda et al. 1992). Receptor tyrosine kinase C-kit is a marker of ICC (Burns et al. 1997;Maeda et al. 1992). ...
... They are considered to be a specialized population of smooth muscle cells embedded in and around the musculature of the GI tract (Burns et al. 1997;Maeda et al. 1992). Receptor tyrosine kinase C-kit is a marker of ICC (Burns et al. 1997;Maeda et al. 1992). ICC play an important role as a pacemaker in the generation of electric slow waves for GI motility (Mostafa et al. 2010; Thomsen et al. 1998). ...
Infections with bovine viral diarrhea virus (BVDV) contribute significantly to health-related economic losses in the beef and dairy industries and are widespread throughout the world. Severe acute BVDV infection is characterized by a gastrointestinal (GI) inflammatory response. The mechanism of inflammatory lesions caused by BVDV remains unknown. The interstitial cells of Cajal (ICC) network plays a pivotal role as a pacemaker in the generation of electrical slow waves for GI motility, and it is crucial for the reception of regulatory inputs from the enteric nervous system. The present study investigated whether ICC were a good model for studying GI inflammatory lesions caused by BVDV infection. Primary ICC were isolated from the duodenum of Merino sheep. The presence of BVDV was detected in ICC grown for five passages after BVDV infection, indicating that BVDV successfully replicated in ICC. After infection with BVDV strain TC, the cell proliferation proceeded slowly or declined. Morphological changes, including swelling, dissolution, and formation of vacuoles in the ICC were observed, indicating quantitative, morphological and functional changes in the cells. RNA sequencing (RNA-Seq) was performed to investigate differentially expressed genes (DEGs) in BVDV-infected ICC and explore the molecular mechanism of underlying quantitative, morphological and functional changes of ICC. Eight hundred six genes were differentially expressed after BVDV infection, of which 538 genes were upregulated and 268 genes were downregulated. Gene Ontology (GO) enrichment and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analyses showed that the 806 DEGs were significantly enriched in 27 pathways, including cytokine-cytokine receptor interaction, interleukin (IL)-17 signaling and mitogen-activated protein kinase (MAPK) signaling pathways. The DEGs and raw files of high-throughput sequencing of this study were submitted to the NCBI Gene Expression Omnibus (GEO) database (accession number GSE122344). Finally, 21 DEGs were randomly selected, and the relative repression levels of these genes were tested using the quantitative real-time PCR (qRT-PCR) to validate the RNA-Seq results. The results showed that the related expression levels of 21 DEGs were similar to RNA-Seq. This study is the first to establish a new infection model for investigating GI inflammatory lesions induced by BVDV infection. RNA-Seq-based transcriptomic profiling can provide a basis for study on BVDV-associated inflammatory lesions.
... We observed that the morphology of ICCs was similar to those in previous reports on the guinea pig small intestine 30 , the human GIT 31 , and chicken oviducts 32 . In previous studies, c-kit protein specifically labeled ICCs in bovines 24 , rats 33 , mice 34 , and guinea pigs 35 . Hudson et al 36 . ...
... Previous studies have reported that ICC-IM were stellate or spindle in shape and existed within the intramuscular layer in the stomachs of guinea pigs 35 and humans 37 . In the present study, the morphology and distribution of ICC-IM are consistent with these reports. ...
The interstitial cells of Cajal (ICCs) are regarded as pacemakers and are involved in neurotransmission in the gastrointestinal tract (GIT) of animals. However, limited information is available about the existence of ICCs within the GIT of ruminants. In this study, we investigated the ultrastructural characteristics and distribution of ICCs in goat abomasum using transmission electron microscopy and c-kit immunohistochemistry. Two different kinds of c-kit immunoreactive cells were observed in the abomasum. The first was identified as ICCs, which appeared to be multipolar or bipolar in shape, with some processes. These c-kit immunoreactive cells were deposited in the submucosal layer, myenteric plexus between the circular and longitudinal muscle layers, and within the longitudinal and circular muscle layers of the abomasum. The second type of cell was round in shape and was identified as mast cells, which were located in the submucosal layer as well as in the lamina propria. Ultrastructurally, ICCs were also observed as stellate or spindle-shaped cells, which were consistent in shape with our c-kit immunoreactive cells. In the cytoplasm of ICCs, numerous mitochondria, rough endoplasmic reticulum, and caveolae were detected. ICCs were located in the myenteric plexus between the longitudinal and circular muscle layers (ICC-MY), with the longitudinal and circular muscle layer was replaced as “intramuscular layers” (ICC-IM), and in the submucosal layer (ICC-SM). In addition, we found ICCs surrounding nerve fibers and smooth muscle cells, where they formed heterocellular junctions in the form of close membrane associations or gap junctions and homocellular junctions among the processes of the ICCs. In the current study, we provide the first complete characterization of ICCs within the goat abomasum and propose that ICCs might have a key role in producing contractions in the ruminant stomach for proper absorption of nutrients.
... The mechanisms underlying contractile activity within the esophagus are complex in that it is composed of both skeletal and smooth muscle with differences in composition occurring between species. In the human esophagus the proximal one third is skeletal muscle, the distal one third is smooth muscle and the middle one third represents a transition from skeletal to smooth muscle (Meyer et al., 1986) Previous studies evaluating the distribution of ICC in the esophagus of the dog, guinea-pig and mouse noted the presence of ICC within the skeletal muscle region (Berezin et al., 1994, Burns et al., 1997, Daniel and Posey-Daniel, 1984, Rumessen et al., 2001) albeit their density was much lower than in the smooth muscle of the adjacent lower esophageal sphincter (LES). Additional studies have characterized the distribution of inhibitory nitrergic and excitatory cholinergic nerve bers within the guinea-pig esophagus, and similar to the distribution of ICC, the greatest density of these nerve bers was within the LES and declined proximally (Brookes et al., 1996). ...
Interstitial cells of Cajal (ICC) and PDGFRα ⁺ cells regulate smooth muscle motility in the gastrointestinal (GI) tract. However, their role(s) in esophageal motility are still unclear. The mouse esophagus has traditionally been described as almost entirely skeletal muscle in nature though ICC have been identified along its entire length. The current study evaluated the distribution of skeletal and smooth muscle within the esophagus using a mouse selectively expressing eGFP in smooth muscle cells (SMCs). The relationship of SMCs to ICC and PDGFRα ⁺ cells was also examined. SMCs declined in density in the oral direction however SMCs represented ~ 25% of the area in the distal esophagus suggesting a likeness to the transition zone observed in humans. ANO1 ⁺ intramuscular ICC (ICC-IM) were distributed along the length of the esophagus though like SMCs, declined proximally. ICC-IM were closely associated with SMCs but were also found in regions devoid of SMCs. Intramuscular and submucosal PDGFRα ⁺ cells were densely distributed throughout the esophagus though only intramuscular PDGFRα ⁺ cells within the LES and distal esophagus highly expressed SK3. ICC-IM and PDGFRα ⁺ cells were closely associated with nNOS ⁺ , VIP ⁺ , VAChT ⁺ and TH ⁺ neurons throughout the LES and distal esophagus. GFAP ⁺ cells resembling intramuscular enteric glia were observed within the muscle and were closely associated with ICC-IM and PDGFRα ⁺ cells, occupying a similar location to motor nerve fibers. These data suggest that the mouse esophagus is more similar to the human than thought previously and thus set the foundation for future functional and molecular studies using transgenic mice.
... Intramuscular interstitial cells (ICC-IM) modulate neurotransmitter responses (10), and myenteric interstitial cells (ICC-MY) serve as pacemakers by generating slow waves that influence smooth muscle contractions (11). On the other hand, submucosal interstitial cells (ICC-SM) coordinate the regulation of secretions and reflexes within the mucosal and submucosal layers (12), while septal interstitial cells (ICC-SEP) function as a supportive network, maintaining the structural integrity of the gastrointestinal wall (13,14). However, as time passes, the complexity and diversity of ICC research have steadily increased, extending to multiple systems, such as the nervous, digestive, and urinary systems, among others (9,15,16). ...
Objective
The significance of interstitial cells of Cajal (ICC) in the gastrointestinal tract has garnered increasing attention. In recent years, approximately 80 articles on ICC have been published annually in various journals. However, no bibliometric study has specifically focused on the literature related to ICC. Therefore, we conducted a comprehensive bibliometric analysis of ICC to reveal dynamic scientific developments, assisting researchers in exploring hotspots and emerging trends while gaining a global perspective.
Methods
We conducted a literature search in the Web of Science Core Collection (WoSCC) from January 1, 2013, to December 31, 2023, to identify relevant literature on ICC. We employed bibliometric software, namely VOSviewer and CiteSpace, to analyze various aspects including annual publication output, collaborations, research hotspots, current status, and development trends in this domain.
Results
A total of 891 English papers were published in 359 journals by 928 institutions from 57 countries/regions. According to the keyword analysis of the literature, researchers mainly focused on “c-Kit,” “expression,” “smooth muscle,” and “nitric oxide” related to ICC over the past 11 years. However, with “SIP syncytium,” “ANO1,” “enteric neurons,” “gastrointestinal stromal tumors (GIST),” and “functional dyspepsia (FD),” there has been a growing interest in the relationship between ANO1, SIP syncytium, and ICC, as well as the role of ICC in the treatment of GIST and FD.
Conclusion
Bibliometric analysis has revealed the current status of ICC research. The association between ANO1, SIP syncytium, enteric neurons and ICC, as well as the role of ICC in the treatment of GIST versus FD has become the focus of current research. However, further research and collaboration on a global scale are still needed. Our analysis is particularly valuable to researchers in gastroenterology, oncology, and cell biology, providing insights that can guide future research directions.
... ICCs, discovered by the Spanish neuroanatomist Santiago Ramón y Cajal, are present around the intermuscular plexus and in the circular muscle layer of the gastrointestinal tract [25]. They have been identified in various parts of the gastrointestinal tract, including the esophagus, stomach, cecum, and small intestine [26,27]. ICCs are thought to act as pacemakers in intestinal peristalsis. ...
Male infertility is a significant factor in approximately half of all infertility cases and is marked by a decreased sperm count and motility. A decreased sperm count is caused by not only a decreased production of sperm but also decreased numbers successfully passing through the male reproductive tract. Smooth muscle movement may play an important role in sperm transport in the male reproductive tract; thus, understanding the mechanism of this movement is necessary to elucidate the cause of sperm transport disorder. Recent studies have highlighted the presence of platelet-derived growth factor receptor α (PDGFRα)-positive interstitial cells (PICs) in various smooth muscle organs. Although research is ongoing, PICs in the male reproductive tract may be involved in the regulation of smooth muscle movement, as they are in other smooth muscle organs. This review summarizes the findings to date on PICs in male reproductive organs. Further exploration of the structural, functional, and molecular characteristics of PICs could provide valuable insights into the pathogenesis of male infertility and potentially lead to new therapeutic approaches.
... Similarly, the muscle tension of isolated gallbladder muscle strips also showed a significant decrease in AC groups ( Figure 3B). CD117/c-Kit protein expression is a specific marker of ICCs [26]. IHC results showed the density of ICCs (labelled with c-Kit) in each AC group (AC12h, AC24h, and AC48h) was extremely reduced (Figure 4). ...
Background:
Impaired interstitial cells of Cajal (ICCs) are central to the pathophysiology of acute cholecystitis (AC). Common bile duct ligation is a common model of AC, producing acute inflammatory changes and decrease in gallbladder contractility.
Aim:
To investigate the origin of slow wave (SW) in the gallbladder and the effect of ICCs on gallbladder contractions during the process of AC.
Methods:
Methylene blue (MB) with light was used to establish selective impaired ICCs gallbladder tissue. Gallbladder motility was assessed using the frequency of SW and gallbladder muscle contractility in vitro in normal control (NC), AC12h, AC24h, and AC48h groups of guinea pigs. Hematoxylin and eosin and Masson-stained gallbladder tissues were scored for inflammatory changes. ICCs pathological changes alterations were estimated using immunohistochemistry and transmission electron microscopy. The alterations of c-Kit, α-SMA, cholecystokinin A receptor (CCKAR), and connexin 43 (CX43) were assessed using Western blot.
Results:
Impaired ICCs muscle strips resulted in the decrease in gallbladder SW frequency and contractility. The frequency of SW and gallbladder contractility were significantly lower in the AC12h group. Compared with the NC group, the density and ultrastructure of ICCs were remarkably impaired in the AC groups, especially in the AC12h group. The protein expression levels of c-Kit were significantly decreased in the AC12h group, while CCKAR and CX43 protein expression levels were significantly decreased in the AC48h group.
Conclusion:
Loss ICCs could lead to a decrease in gallbladder SW frequency and contractility. The density and ultrastructure of ICCs were clearly impaired in the early stage of AC, while CCKAR and CX43 were significantly reduced at end stage.
... These specific staining techniques led to the previous assumption that ICCs are primitive neurons, as they were unable to truly discriminate between neurons and ICCs. [63,64], and guinea-pigs [65,66]. These studies and further investigations have increased our understanding of the complex architecture of ICC networks in relation to the ENS and the intestinal smooth muscle layers [67]. ...
Interstitial cells of Cajal (ICCs) are pacemaker cells of gastrointestinal motility that generate and transmit electrical slow waves to smooth muscle cells in the gut wall, thus inducing phasic contractions and coordinated peristalsis. Traditionally, tyrosine-protein kinase Kit (c-kit), also known as CD117 or mast/stem cell growth factor receptor, has been used as the primary marker of ICCs in pathology specimens. More recently, the Ca ²⁺ -activated chloride channel, anoctamin-1, has been introduced as a more specific marker of ICCs. Over the years, various gastrointestinal motility disorders have been described in infants and young children in which symptoms of functional bowel obstruction arise from ICC-related neuromuscular dysfunction of the colon and rectum. The current article provides a comprehensive overview of the embryonic origin, distribution, and functions of ICCs, while also illustrating the absence or deficiency of ICCs in pediatric patients with Hirschsprung disease intestinal neuronal dysplasia, isolated hypoganglionosis, internal anal sphincter achalasia, and congenital smooth muscle cell disorders such as megacystis microcolon intestinal hypoperistalsis syndrome.
... In the gastrointestinal tract, Interstitial Cells of Cajal (ICCs) expressing c-Kit receptor tyrosine kinase serve as pacemaker cells and mediators of neuromuscular transmission [32,33]. Reduced or absent ICC has been found in the colon of constipated patients [34]. ...
A vital bioactive component of marine resources is Holothuria leucospilota polysaccharides (HLP). This study examined whether HLP could regulate intestinal flora to treat loperamide-induced constipation. Constipated mice showed signs of prolonged defecation (up by 60.79 min) and a reduced number of bowel movements and pellet water content (decreased by 12.375 and 11.77%, respectively). The results showed that HLP treatment reduced these symptoms, reversed the changes in related protein expression levels in the colon, and regulated the levels of active peptides associated with the gastrointestinal tract in constipated mice, which significantly improved water-electrolyte metabolism and enhanced gastrointestinal motility. Meanwhile, it was found that intestinal barrier damage was reduced and the inflammatory response was inhibited through histopathology and immunohistochemistry. As a means to further relieve constipation symptoms, treatment with low, medium, and high HLP concentrations increased the total short-chain fatty acid (SCFA) content in the intestine of constipated mice by 62.60 μg/g, 138.91 μg/g, and 126.51 μg/g, respectively. Moreover, an analysis of the intestinal flora’s gene for 16S rRNA suggested that the intestinal microbiota was improved through HLP treatment, which is relevant to the motivation for the production of SCFAs. In summary, it was demonstrated that HLP reduced loperamide-induced constipation in mice.
... Compared to the constipated control group without MOS treatment, the ICC count in the MOS treated groups was significantly higher (Figure 7, P < 0.01 and P < 0.001, respectively). ICC plays a pivotal role in controlling intestinal motility and is present in all layers of the colon [15,35]. A decrease in ICC leads to slow bowel movements and decreased smooth muscle contractile activity, which can cause constipation. ...
Constipation is a chronic disease caused by infrequent, inadequate, and difficult bowel movements. The present study aimed to evaluate the potential laxative effect of maltooligosaccharide (MOS) on loperamide-induced constipation in a rat model. In vitro experiments were conducted to evaluate the effect of MOS on the growth of lactic acid bacteria. Moreover, to examine the effect of MOS administration on Sprague-Dawley (SD) rats with loperamide-induced constipation, the drinking water for the rats was supplemented with 10% or 15% of MOS for 14 days, and, thereafter, the improvement in constipation was assessed. For this, the rats were divided into five groups: normal (Nor), loperamide-induced constipated (Con), positive control (15% of dual-oligosaccharide (DuO-15)), 10% MOS treated (MOS-10), and 15% MOS-treated (MOS-15). In an in vitro test, MOS treatment promoted the growth of lactic acid bacteria except Lactobacillus bulgaricus. Treatment with higher MOS dose relieved constipation in rats by improving the fecal pellet and water content. Furthermore, in the high MOS dose group, the cecal short-chain fatty acid levels significantly increased compared to those in the control group (P
... Pacemaker ICC in these regions are coupled electrically to one another and to adjacent smooth muscle cells (SMCs) via gap junctions allowing conduction of SWs from ICC to SMCs where excitation-contraction coupling occurs 15 . These cells are typically highly-branched stellate-shaped cells 16,17 . In contrast, another population of spindle-shaped intramuscular ICC (ICC-IM) are involved in neuromuscular transmission 16,18 . ...
The internal anal sphincter (IAS) generates phasic contractions and tone. Slow waves (SWs) produced by interstitial cells of Cajal (ICC) underlie phasic contractions in other gastrointestinal regions. SWs are also present in the IAS where only intramuscular ICC (ICC-IM) are found, however the evidence linking ICC-IM to SWs is limited. This study examined the possible relationship between ICC-IM and SWs by recording Ca²⁺ transients in mice expressing a genetically-encoded Ca²⁺-indicator in ICC (Kit-Cre-GCaMP6f). A role for L-type Ca²⁺ channels (CavL) and anoctamin 1 (ANO1) was tested since each is essential for SW and tone generation. Two distinct ICC-IM populations were identified. Type I cells (36% of total) displayed localised asynchronous Ca²⁺ transients not dependent on CavL or ANO1; properties typical of ICC-IM mediating neural responses in other gastrointestinal regions. A second novel sub-type, i.e., Type II cells (64% of total) generated rhythmic, global Ca²⁺ transients at the SW frequency that were synchronised with neighbouring Type II cells and were abolished following blockade of either CavL or ANO1. Thus, the spatiotemporal characteristics of Type II cells and their dependence upon CavL and ANO1 all suggest that these cells are viable candidates for the generation of SWs and tone in the IAS.
... They can be identified by expression of glial fibrillary acidic protein (GFAP), an intermediate filament involved in controlling the shape of glial cells, and S-100, a protein involved in the stimulation of neurons [2,5]. Finally, the ICC are commonly identified by expression of c-kit, a cell signaling molecule; however, c-kit is also present in a variety of cell types [6]. ...
... ICC-MY are not present in the fundus. [97] In all regions of the stomach, ICC can be found inside the longitudinal and the circumferential muscle layers, so called intramuscular ICC (ICC-IM). In addition to ICC, also platelet-derived growth factor receptor í µí»¼ posi- tive (PDGFRí µí»¼ + ) cells are located at the myenteric plexus and inside the muscle layers. ...
Mathematical and computational modeling of the stomach is an emerging field of biomechanics where several complex phenomena, such as gastric electrophysiology, fluid mechanics of the digesta, and solid mechanics of the gastric wall, need to be addressed. Developing a comprehensive multiphysics model of the stomach that allows studying the interactions between these phenomena remains one of the greatest challenges in biomechanics. A coupled multiphysics model of the human stomach would enable detailed in‐silico studies of the digestion of food in the stomach in health and disease. Moreover, it has the potential to open up unprecedented opportunities in numerous fields such as computer‐aided medicine and food design. This review article summarizes our current understanding of the mechanics of the human stomach and delineates the challenges in mathematical and computational modeling which remain to be addressed in this emerging area.
... They initiate and propagate electrical slow waves, which regulate gut peristalsis and mediate neurotransmission between the enteric neurons and smooth muscle of the bowel (Burns, 2007). ICC are found throughout the entire gastrointestinal tract in several distinct populations with different functional roles (Komuro, 2006, Burns et al., 1997. The most essential populations are the branching ICC networks associated with the myenteric plexus, which generates spontaneous slow rhythmical pacemaker activity (Hirst et al., 2006), and the spindle shaped ICC located intramuscularly that propagate electrical waveforms throughout the muscle (Huizinga and Chen, 2014). ...
BACKGROUND: Gastroschisis is a paraumbilical abdominal wall defect through which the bowel herniates. The bowel is in direct contact with the amniotic fluid. At birth the bowel is often thickened and shortened. The main gastroschisis-related morbidity is that of intestinal dysmotility but the mechanism(s) underlying this are poorly understood and no treatment advances have occurred since the advent of parenteral nutrition. Human studies have shown that the third trimester amniotic fluid of gastroschisis pregnancies is proinflammatory. In addition, in animal models of gastroschisis and human pathological gut tissue interstitial cells of Cajal (ICC – pacemaker cells of the gut) have been found to be decreased in number and immature within gastroschisis bowel. HYPOTHESES: Gastroschisis-related intestinal dysfunction (GRID) is secondary to deficient, immature bowel wall ICC. AIMS: My primary aim in this thesis was to investigate the development of ICC in gastroschisis utilising genetic animal models and human pathological gut tissue. Additionally, I aimed to determine the overall morphology of gastroschisis bowel wall, the impact of inflammatory modulation on bowel wall development and the impact of clinical antenatal interventions on infant outcomes. METHODS: Quantitative analysis of bowel wall ICC, enteric neurons and architecture were performed utilising genetic animal gastroschisis models and human pathological tissue. Modulation of in-utero inflammation was performed through in-utero injections of IL-8 (pro-inflammatory protein) in a genetic animal gastroschisis model. Finally, retrospective clinical data on gastroschisis were collected from three paediatric surgical centres to determine whether early delivery or administration of maternal antenatal corticosteroids could improve infant morbidity. RESULTS: Phenotypic analysis of the Scribble knockout mouse model revealed the expressed abdominal wall defect (AWD) to be that of exomphalos rather than gastroschisis. Additionally, phenotypic analysis of the aortic carboxypeptidase-like protein (ACLP) knockout mouse showed the AWD was not characteristic of gastroschisis as the externalised bowel was free floating within exocoelomic fluid and not the amniotic fluid. Bowel wall ICC were architecturally normal but reduced in number in AWD fetuses compared to controls. In-utero injection of IL-8 further reduced ICC numbers. In contrast, analysis of human pathological gut tissue showed no difference in the number or architecture of bowel wall ICC between gastroschisis and control tissue. However, the gastroschisis bowel wall was significantly thicker than that of controls with evidence of smooth muscle hyperplasia and deficiency of α-smooth muscle actin within the longitudinal muscle layer. The clinical retrospective data showed that early delivery of gastroschisis fetuses did not improve bowel function and was associated with prolonged time to full enteral feeds and length of hospital stay. Additionally, there was no evidence that administration of maternal antenatal corticosteroids improved time to full enteral feeds or length of hospital stay in gastroschisis infants. CONCLUSION: Phenotypic analysis of abdominal wall defects in murine models is challenging. However, accurate delineation of the defect anatomy is essential to ensure appropriate result reporting and data analysis. The presented data suggests the ICC may be less important as the cause of GRID than originally expected. However, another potential cause for GRID is bowel wall thickening comprising of smooth muscle hyperplasia and possible bowel myopathy for which there are several hypothesised triggers. Additionally, the data presented in this thesis do not support early delivery of gastroschisis fetuses or the administration of maternal antenatal corticosteroids as methods to improve gastroschisis infant outcomes. The results of this thesis have generated some important findings and have highlighted a number of novel hypotheses for the trigger of GRID. Further research on the basis of these findings within an appropriate animal model and through prospective non-invasive human studies are required in order to develop a targeted antenatal therapy to improve gastroschisis infant bowel function and clinical outcomes.
... Escherichia coli is among the first bacterial species to colonise in intestine during infancy [14]. On the basis of their virulence and disease manifestation, there are five distinct groups of E. coli, which include toxin-producing strains like enterotoxigenic (ETEC), enterohaemorrhagic (EHEC) or verocytotoxigenic E. coli (VTEC), enteroaggregative (EAggEC), non-toxic strains like enteropathogenic (EPEC) and enteroinvasive (EIEC) E. coli [3]. These groups are associated with diarrhoea, haemorrhagic colitis (HC), dysentery, bladder and kidney infections, surgical wound infection, septicaemia, haemolytic uraemic syndrome (HUS), pneumonia and meningitis, and some of these conditions result in death. ...
The aim of this study was comparative evaluation of loop-mediated isothermal amplification (LAMP) and polymerase chain reaction (PCR) assay for rapid and inexpensive detection of shiga toxin-producing E. coli in animal origin foods by targeting stx1 and stx2 genes. The LAMP assay was performed using a water bath. The standardized LAMP assay was evaluated on 122 E. coli field isolates obtained from various animal origin food samples to ensure its reliability and usefulness. The result showed that conventional PCR could detect 68 (55.73%) and 75 (61.47%) positive E. coli isolates for stx1 and stx2 genes. Whereas, LAMP showed higher sensitivity by detecting 79 (64.75%) and 87 (71.31%) positive isolates of E. coli for stx1 and stx2 genes, respectively. LAMP assay was found to be highly specific and 10 times more sensitive as it could detect 1.11 × 10² cfu/ml for both stx1 and stx2 genes of E. coli isolates, whereas conventional PCR could detect 1.85 x 10³ cfu/ml for both stx1 and stx2 genes of E. coli isolates. The rapidness, sensitivity, specificity, easiness and cost-effectiveness of LAMP assays will be very useful for the detection of foodborne pathogens for improving food sanitation and maintaining food safety.
... Pressure induced by an existing patch of HFLAC may activate either longitudinal or circumferential neural or myogenic mechanoreceptors in a relaxed area setting up a resonance effect. The vectoring of the tension through the wall will depend on the orientation of the fibers through which it is conducted as well as the situation of the relaxed area in relation to the shape of the cavity and hence may be expected to engender either a longitudinally or circumferentially orientated subsequent patch of HFLAC given that both longitudinal and circumferential muscle layers of the fundus contain ICC-IM (11). This hypothesis is supported by the fact that, at any given time, a number of patches were seen in the clamped fundus each with either radially or circumferentially propagating contractions (Fig. 6) as opposed to the broadly confluent patches of radial and circumferential contraction that were seen in the unclamped corpus. ...
We used spatiotemporal mapping of strain rate to determine the direction of propagation and amplitudes of the longitudinal and circumferential components of antro-corporal contractions and fundal contractions in the rat stomach maintained ex vivo and containing a volume of fluid that was within its normal functional capacity. In the region of the greater curvature the longitudinal and circular components of anterocorporal (AC) contractions propagated synchronously at right angles to the arciform geometric axis of the stomach. However the configuration of AC contractions was u shaped, neither the circular nor the longitudinal component of contractions being evident in the upper proximal corpus. Similarly, in the distal upper antrum of some preparations circumferential components propagated more rapidly than longitudinal components. Ongoing 'high frequency, low amplitude myogenic contractions' i.e. HFLAC were identified in the upper proximal gastric corpus and on the anterior and posterior wall of the fundus. The amplitudes of these contractions were modulated in the occluded stomach by low frequency pressure waves which occurred spontaneously. Hence the characteristics of phasic contractions vary regionally in the antrum and corpus and a previously undescribed high frequency contractile component was identified in the proximal corpus and fundus, the latter being modulated in synchrony with cyclic variation in intrafundal pressure in the occluded fundus.
... These observations are consistent with ICC expression throughout the GI tract where they are able to coordinate contraction and are key in regulating gut motility patterns. Physiological studies have shown that mice with defective c-Kit signaling, which is critical for ICC rhythmicity, have a reduced contractile response after neural stimulation and GI motility defects (Ward et al. 1994, Burns et al. 1997, Beckett et al. 2007). The ENS and ICC represent two well-studied examples of the tight coordination required across multiple cell types to produce rhythmic GI motility. ...
Coordinated smooth muscle contraction is critical for force production and proper functioning of numerous organ systems. Activation at the myosin motor domain via phosphorylated myosin light chain (phospho-MLC) remains the primary signal to initiate contraction, but it is now appreciated that there are additional force modulators also present in smooth muscle. One particularly well studied modulatory protein is Caldesmon (CaD), which has been implicated in controlling contractile force in vascular smooth muscle, however little is known of CaD's physiological role in vivo. Studies in vitro have shown that CaD inhibits actomyosin interactions and that this effect is reversed after phosphorylation, allowing for greater force propagation. Since a number of gastrointestinal (GI) tract and vascular disorders are known to be a result of aberrant force production, closely monitoring CaD's functional properties may provide insight into common contractile defects. We took advantage of the transparent nature of the intestine in larval zebrafish to study CaD's effect on smooth muscle contraction in a vertebrate model. We initiated these studies by examining propulsive peristalsis in the larval intestine after knockdown of endogenous smooth muscle CaD protein. We next measured the role of CaD in the absence of phospho-MLC to better understand its function in disease states where myosin activation is perturbed. Using extensive live imaging analysis, we show that disrupting CaD function within intestinal smooth muscle can significantly increase GI motility, with and without phospho-MLC, highlighting CaD's ability to independently modulate contractile force. In addition, previous work on a mutant, meltdown (mlt), in our lab has uncovered a smooth muscle myosin (myh11) mutation leading to increased contractile force and premature CaD phosphorylation. Interestingly, in the mlt mutant intestinal epithelial invasion was observed pointing to the unique role for force propagation in initiating cell invasion. We show that CaD is necessary for mlt epithelial invasion to occur, as knockdown of CaD causes the invasive phenotype in heterozygous mlt, which otherwise appear wild type. To gain a better understanding of the crosstalk between muscle contraction and epithelial invasion, we performed a genetic screen for modifier mutants of the mlt phenotype. From the screen, we discovered two enhancer mutants of mlt that contained missense mutations in unique protein domains of MYH11 that alter the contractile function of smooth muscle. These mutations (S237Y and L1287M) occur in both the motor domain and helical tail domain of the protein, suggesting that alterations in distinct regions of myosin can result in abnormal contraction and potentially lead to invasion in underlying cells. Since a number of myosin mutations have been implicated in vascular disease and colon cancer, these studies provide insight into the diversity and mechanistic consequences of mutated myosin in altering smooth muscle contraction and epithelial cell invasion.
... Chacun des deux types cellulaires semble être retrouvé dans des régions précises dans les tissus des organes [341,358]. Ainsi, les cellules multipolaires sont retrouvées au niveau du plexus myentérique de l'intestin, c'est-à-dire entre les deux couches de muscles circulaires et longitudinales ainsi que dans la muqueuse et la sous-muqueuse du colon. En revanche, les cellules allongées sont préférentiellement retrouvées dans la couche musculaire plus profonde. ...
Atrial fibrillation (AF) is the most common arrhythmia encountered in clinical practice. As its pathophysiology is still poorly understood, it is difficult to treat. Several parameters have been described as involved in the initiation of atrial fibrillation, but the precise mechanisms of initiation of atrial fibrillation are not understood. In this study, histological, biochemical, genetic and transcriptomic approaches have been performed in order to identify the substrates involved in the initiation of human atrial fibrillation. Thus, it has been demonstrated presence of interstitial cells Cajal-like in myocardial sleeves of pulmonary veins that may be the cause of ectopic foci, a possible early embryonic origin through lack of expression of the pitx2 gene in atrial fibrillation patients, overexpression of the autonomic nervous system to both adrenergic and cholinergic pathways, a change in the flow of potassium current IKs through intervention of KCNE regulatory proteins and cytoskeletal muscle protein. In addition, a complication of atrial fibrillation is the occurrence of stroke. It has been shown at left atrial level an overexpression of Von Willebrand factor in patients with atrial fibrillation and an increase of serological VEGF in paroxysmal subtype of atrial fibrillation. These new data allow completing the knowledge on atrial fibrillation and subsequently considering the possibility of new therapeutic strategies that could be more effective.
... The characteristic ultrastructure of ICC is the existence of caveolae, intermediate filaments, well-developed mitochondria, a basal lamina, and large gap junctions between cells. The c-Kit receptor tyrosine kinase, whose natural ligand is stem cell factor (SCF), is expressed on the cell surface of ICC, and is an established marker for immunohistochemical identification of these cells (9,10). ...
Interstitial cells of Cajal (ICC) are mesenchymal cells that are distributed along the gastrointestinal tract and function as pacemaker cells or intermediary cells between nerves and smooth muscle cells. ICC express a receptor tyrosine kinase c-Kit, which is an established marker for ICC. The c-kit gene is allelic with the murine white-spotting locus (W), and some ICC subsets were reported to be missing in heterozygous mutant W/W(v) mice carrying W and W(v) mutated alleles. In this study, the characterization of interstitial cells in the subserosal layer of W/W(v) mice was analyzed by immunohistochemistry and electron microscopy. In the proximal and distal colon of W/W(v) mutant mice, no c-Kit-positive cells were detected in the subserosal layer by immunohistochemistry. By electron microscopy, the interstitial cells, which were characterized by the existence of caveolae, abundant mitochondria and gap junctions, were observed in the W/W(v) mutant colon. The morphological characteristics were comparable to those of the multipolar c-Kit positive ICC seen in the subserosa of proximal and distal colon of wild-type mice. Fibroblasts were also located in the same layers, but the morphology of the fibroblasts was distinguishable from that of ICC in wild type mice or of ICC-like cells in W/W(v) mutant mice. Collectively, it is concluded that c-Kit-negative interstitial cells showing a typical ICC ultrastructure exist in the proximal and distal colon of W/W(v) mutant mice.
... 5 There are at least 2 distinct classes of ICC in the stomach. ICC-MY lie in the plane of the myenteric plexus between the circular and longitudinal muscle layers, 7,8 and intramuscular ICC (ICC-IM) lie within muscle bundles throughout most of the stomach. 9 However, examination of antral longitudinal muscles of some species shows few ICC-IM in this region. ...
Gastric peristalsis begins in the orad corpus and propagates to the pylorus. Directionality of peristalsis depends upon orderly generation and propagation of electrical slow waves and a frequency gradient between proximal and distal pacemakers. We sought to understand how chronotropic agonists affect coupling between corpus and antrum.
Electrophysiological and imaging techniques were used to investigate regulation of gastric slow wave frequency by muscarinic agonists in mice. We also investigated the expression and role of cholinesterases in regulating slow wave frequency and motor patterns in the stomach.
Both acetycholinesterase (Ache) and butyrylcholine esterase (Bche) are expressed in gastric muscles and AChE is localized to var-icose processes of motor neurons. Inhibition of AChE in the absence of stimulation increased slow wave frequency in corpus and throughout muscle strips containing corpus and antrum. CCh caused depolarization and increased slow wave frequency. Stimulation of cholinergic neurons increased slow wave frequency but did not cause depolarization. Neostigmine (1 μM) in-creased slow wave frequency, but uncoupling between corpus and antrum was not detected. Motility mapping of contractile activity in gastric muscles showed similar effects of enteric nerve stimulation on the frequency and propagation of slow waves, but neostigmine (> 1 μM) caused aberrant contractile frequency and propagation and ectopic pacemaking.
Our data show that slow wave uncoupling is difficult to assess with electrical recording from a single or double sites and sug-gest that efficient metabolism of ACh released from motor neurons is an extremely important regulator of slow wave frequency and propagation and gastric motility patterns.
... Chacun des deux types cellulaires semble être retrouvé dans des régions précises dans les tissus des organes [341,358]. Ainsi, les cellules multipolaires sont retrouvées au niveau du plexus myentérique de l'intestin, c'est-à-dire entre les deux couches de muscles circulaires et longitudinales ainsi que dans la muqueuse et la sous-muqueuse du colon. En revanche, les cellules allongées sont préférentiellement retrouvées dans la couche musculaire plus profonde. ...
... 97 Finally, the ICC are identified by expression of c-kit, a cell signaling molecule; however, c-kit is also present in a variety of cell types. 98 Extensive investigations were performed by our group on the ovine esophagus to determine the location of the ganglion cells within the structure of the esophagus (►Fig. 11). ...
Although being a tubular structure, the esophagus is an extremely complex organ to engineer. To engineer an organ, its components and their structure and function must be well understood. With regards to esophagus, extensive investigations have been performed in experimental models to understand the nature of the esophageal epithelial cells with regards to their isolation, culture, and growth on scaffolds to generate epithelium. Special subpopulations of these cells have been identified that possess proliferative capabilities with subsequent differentiative capacity to generate epithelium. Studies have also been performed to obtain data on the possibilities of utilizing esophageal biopsies from esophagus damaged after caustic exposure for tissue engineering applications. Subsequently, attention is being paid to the esophageal smooth muscle which is an extremely complex structure responsible for the propulsive activities. In addition to the muscle complex, proper functioning of the esophagus will require understanding of the enteric nervous system (ENS) that controls the propulsive activity in a coordinated manner. Investigations have been performed to better understand the esophageal ENS and to isolate and maintain these cells under tissue culture conditions. Besides the cellular elements, studies have also been performed to seed these cells on scaffolds and study the constructs with regards to cell attachment and viability under tissue culture conditions. Tests have also been performed on native esophageal tissue to understand the functioning of this tissue under the effect of pharmacological agents and to establish norms to compare engineered esophageal tissue. Vascularization, which is a limiting factor in tissue engineering, has been approached with the in situ bioreactor concept using the omentum not only to provide vascular ingrowth but also to offer a pedicle for the engineered esophagus to enable its surgical transposition. This review offers an insight into the advances in esophagus tissue engineering in a large experimental model using the "hybrid construct" approach which advocates the precise engineering of the tubular gastrointestinal organs based on growth of specific cells on specially designed scaffolds and amalgamating them to create the desired complex tissue structure.
... ICC-IM were more dispersed in adults animals than in juvenile stages. The shape and location of ICC-MP were comparable to ICC-MP in the distal stomach of guinea pigs (Burns et al. 1997 of the lesser curvature regulates inhibitory neurotransmission (Burns et al. 1996). We speculate that the same action can be accomplished by the omasum, because its strong and prolonged contractions have a rate of motor activity independent of the reticulum-rumen and are inhibited by distension of the abomasum (Sellers and Stevens 1966). ...
The interstitial cells of Cajal (ICC) have been reported to regulate gastrointestinal motility. We investigated the distribution and the morphological and morphometric characteristics of the immunohistochemical reaction against c-kit in the forestomachs of fetal, newborn and adult cows. The anti-c-kit reaction revealed different populations of ICC among age groups and organs. ICC were more numerous and smaller in fetuses. Larger ICC were identified in newborns, except for those in the rumen. During the earliest stages of development, ICC were abundant in the inner layer of the muscularis and were consistently associated with this layer. In all samples, ICC were found in the outer layer of the tunica muscularis. ICC were found between the two muscle layers in the omasum at all ages; however, they were identified only in the rumen of the adult. Our study demonstrated that ICC are present in the forestomach of bovines.
... Trendelenburg, in 1917, already recognized that the rhythmic peristaltic activity induced by distention in the guinea pig small intestine can proceed without a nervous conduction system (Trendelenburg, 2006). Slow waves in the guinea pig intestine are likely originating in networks of ICC, which are prominent and appear similar to other animal models (Zhou and Komuro, 1992;Burns et al., 1997;Lavin et al., 1998;Seki et al., 1998). ...
In summary, a number of studies have been published on the mechanisms underlying the segmentation motor pattern. Segmentation in all cases is observed as short-lived contractions that are propagating over a few mm, are often rhythmic in nature involving the whole intestine for a relatively long period or form clusters of segmental contractions for short periods. There is overwhelming evidence that the rhythmicity of segmentation is associated with ICC activity and recently, the classical segmentation motor pattern has been shown to occur in the presence of nerve conduction blockade. However, under most experimental conditions, and in vivo, the enteric nervous system provides an essential stimulus for the motor activity to develop and hence a variety of nerve conduction blockers or neural receptor blockers will inhibit segmentation activity. Whether or not a motor pattern occurs in response to nutrients or luminal distention is often determined by the response of the ENS to the stimulus, including sensory and motor neurons. This is also the case for segmentation, and several components of the ENS have been shown to be involved. This neural activity then works in concert with the ICC pacemaker activities to generate the motor pattern of segmentation.
... The identification of the expression of the gene product c-kit in ICCs was a major breakthrough in ICCs research. C-kit is a protooncogene that encodes the receptor tyrosine kinase kit, which is expressed in ICCs and mast cells within the gastrointestinal tract [35][36][37][38][39]. Consequently many studies have been performed showing the expression of c-kitpositive ICCs in the gastrointestinal tract of several species, including humans [40][41][42][43], mice [44], rats [45,46], and guinea pigs [47,48]. These studies and further investigations have increased our understanding of the complex architecture of ICCs networks in relation to the ENS and the intestinal smooth muscles [49]. ...
Hirschsprung disease (HD) is the most prevalent congenital gastrointestinal motility disorder. The pathogenesis of HD is defined as a functional intestinal obstruction resulting from a defect in the intrinsic innervation of the distal bowel. In addition to the enteric nervous system, the interstitial cells of Cajal (ICC) play an important role in the generation of coordinated gastrointestinal peristalsis. The major function of the ICCs is the generation of slow waves that allow these cells to act as specialised pacemaker cells within various tissues. ICCs have additional functions in the gastrointestinal tract as regulators of mechanical activity and neurotransmission. Due to the central role of ICCs in gastrointestinal peristalsis, it has been suggested that defects or impairments of the ICCs may contribute to motility dysfunction in several gastrointestinal motility disorders. This review describes the distribution and functions of ICCs in the normal gut and in Hirschsprung disease.
The gastrointestinal (GI) tract displays multiple motor patterns that move nutrients and wastes through the body. Smooth muscle cells (SMCs) provide the forces necessary for GI motility, but interstitial cells, electrically coupled to SMCs, tune SMC excitability, transduce inputs from enteric motor neurons and generate pacemaker activity that underlies major motor patterns, such as peristalsis and segmentation. The interstitial cells regulating SMCs are interstitial cells of Cajal (ICC) and PDGFRa+ cells. Together these cells form the SIP syncytium. ICC and PDGFRa+ cells express signature Ca2+-dependent conductances: ICC express Ca2+-activated Cl- channels, encoded by Ano1, that generate inward current, and PDGFRa+ cells express Ca2+-activated K+ channels, encoded by Kcnn3, that generate outward current. The open probabilities of interstitial cell conductances are controlled by Ca2+ release from the endoplasmic reticulum. The resulting Ca2+ transientsoccur spontaneously in a stochastic manner. Ca2+ transients in ICC induce spontaneous transient inward currents and spontaneous transient depolarization (STDs). Neurotransmission increases or decreases Ca2+transients, and the resulting depolarizing or hyperpolarizing responses conduct to other cells in the SIP syncytium. In pacemaker ICC, STDs activate voltage-dependent Ca2+ influx, which initiates a cluster of Ca2+ transients and sustains activation of ANO1 channels and depolarization during slow waves. Regulation of GI motility has traditionally been described as neurogenic and myogenic. Recent advances in understanding Ca2+ handling mechanisms in interstitial cells and how these mechanisms influence motor patterns of the GI tract, suggest the term myogenic should be replaced by the term, SIPgenic, as this review discusses.
The motility of the gallbladder (GB) involves the storage, concentration and delivery of bile. GB motor functions are controlled by multiple complex factors, such as extrinsic and intrinsic innervation, humoral factors and neuropeptides. GB emptying results from coordinated contractions of the muscular layers of the GB wall. Depolarization of GB smooth muscle (GBSM) depends on the activation of the regular depolarization‑repolarization potential, referred to as slow waves (SWs). These rhythmic SWs of GBSM contraction are mediated by several cell types, including smooth muscle cells (SMCs), GB neurons, telocytes (TC) and specialized pacemaker cells called interstitial cells of Cajal (ICC). The present article introduced a new GB motor unit, the SMC‑TC‑ICC‑neuron (STIN) syncytium. In GB, STIN cells provide pacemaker activity, propagation pathways for SWs, transduction of inputs from motor and sensory neurons and mechanosensitivity. The present review provided an overview of STIN cells, mechanisms generating GBSM contractile behavior and GB motility, and discussed alterations of STIN cell function under different disease conditions.
Background
Peripheral corticotropin‐releasing factor (CRF) has been reported to affect gastrointestinal motility through corticotropin‐releasing factor receptor located in enteric nervous system (ENS), but less is known about of the relationship between peripheral CRF and interstitial cells of Cajal (ICC).
Methods
Mice were intraperitoneally injected with CRF receptor agonists to determine their effects on colonic ICC. Chronic heterotypic stress (CHeS) was applied to mice to determine endogenous CRF‐CRF receptor signaling on colonic ICC.
Results
We found that stressin1, a selective CRF receptor 1 (CRF1) agonist, significantly increased the expression of CRF1 but had no effect on the expression of CRF2 in the smooth muscles of murine colon. The protein expression of c‐Kit, Anoctamin‐1 (ANO1), and stem cell factor (SCF) in the colonic smooth muscles was significantly decreased in stressin1‐treated mice. Accordingly, 2‐(4‐Chloro‐2‐methylphenoxy)‐N′‐(2‐methoxybenzylidene) acetohydrazide (Ani 9), a selective ANO1 blocker, had a less significant inhibitory effect on CMMC in stressin1‐treated mice compared to the saline‐treated ones. Similarly, we also found that ICC and ANO1 were reduced in the colonic smooth muscles of mice by treatment with sauvagine (ip), a CRF2 agonist. However, different with stressin1, sauvagine decreased the expression of CRF2 besides increasing CRF1 expression in the colonic smooth muscles. Similar results of CRF1 and c‐Kit expressions were also obtained from the colon of CHeS‐treated mice.
Conclusion
All these results suggest that CRF may be involved in the abnormality of colonic motility through peripheral CRF1 to decrease the number and function of ICC, which provides a potential target for treating stress‐induced gastrointestinal motility disorder.
Introduction:
The network of Interstitial Cells of Cajal (ICC) plays a plethora of key roles in maintaining, coordinating, and regulating the contractions of the gastrointestinal (GI) smooth muscles. Several GI functional motility disorders have been associated with ICC degradation. This study extended a previously reported 2D morphological analysis and applied it to 3D spatial quantification of three different types of ICC networks in the distal stomach guided by confocal imaging and machine learning methods. The characterization of the complex changes in spatial structure of the ICC network architecture contributes to our understanding of the roles that different types of ICC may play in post-prandial physiology, pathogenesis, and/or amelioration of GI dsymotility- bridging structure and function.
Methods:
A validated classification method using Trainable Weka Segmentation was applied to segment the ICC from a confocal dataset of the gastric antrum of a transgenic mouse, followed by structural analysis of the segmented images.
Results:
The machine learning model performance was compared to manually segmented subfields, achieving an area under the receiver-operating characteristic (AUROC) of 0.973 and 0.995 for myenteric ICC (ICC-MP; n = 6) and intramuscular ICC (ICC-IM; n = 17). The myenteric layer in the distal antrum increased in thickness (from 14.5 to 34 μm) towards the lesser curvature, whereas the thickness decreased towards the lesser curvature in the proximal antrum (17.7 to 9 μm). There was an increase in ICC-MP volume from proximal to distal antrum (406,960 ± 140,040 vs. 559,990 ± 281,000 μm3; p = 0.000145). The % of ICC volume was similar for ICC-LM and for ICC-CM between proximal (3.6 ± 2.3% vs. 3.1 ± 1.2%; p = 0.185) and distal antrum (3.2 ± 3.9% vs. 2.5 ± 2.8%; p = 0.309). The average % volume of ICC-MP was significantly higher than ICC-IM at all points throughout sample (p < 0.0001).
Conclusions:
The segmentation and analysis methods provide a high-throughput framework of investigating the structural changes in extended ICC networks and their associated physiological functions in animal models.
The aim of this study was to investigate the effect of a mixture of multi-strain probiotics and prebiotics on loperamide-induced constipation in Sprague-Dawley rats. A multi-strain probiotics alone (loperamide-induced group with multi-strain probiotics mixture group; Lop-Pro) and a mixture of multi-strain probiotics and prebiotics (loperamide-induced group with multi-strain probiotics and prebiotics mixture group; Lop-Pro/Pre) were administered orally after inducing constipation. The fecal water content was significantly higher (by 42%) in the Lop-Pro/Pre group (33.5%) than in the loperamide-induced group (Lop) (23.7%) (p<0.05). The intestinal mucosal thickness, crypt cell area, and interstitial cells of Cajal area were significantly higher in the Lop-Pro/Pre group compared to the Lop group by 16.4%, 20.6%, and 42.3%, respectively. Additionally, the total short-chain fatty acid content was significantly increased in the Lop-Pro and Lop-Pro/Pre groups by 56.4% and 54.2%, respectively, compared with the Lop group. The Lop-Pro and Lop-Pro/Pre groups recovered loperamide-induced alteration in Bacteroidetes and Verrucomicrobia abundance among intestinal microbiota, whereas the Lop-Pro/Pre group recovered Akkermansia, Lactobacillus, Clostridium, Bacteroides, and Oscillibacter abundance. Moreover, the relative abundance of Oscillibacter and Clostridium was significantly different in the Lop-Pro/Pre group compared to the Lop group. Collectively, administration of synbiotics rather than multi-strain probiotics alone is effective in alleviating constipation.
Background
Intrinsic primary afferent neurons (IPANs) enable the gut to manifest reflexes in the absence of CNS input. PKG1α is selectively expressed in a subset of neurons in dorsal root ganglia (DRG) and has been linked to nociception and long‐term hyperexcitability.
Methods
We used immunoblotting, immunocytochemistry, and in vitro assays of IPAN‐dependent enteric functions to test hypotheses that subsets of primary neurons of the ENS and DRG share a reliance on PKG1α expression.
Key Results
PKG1α immunoreactivity was demonstrated in immunoblots from isolated myenteric ganglia. PKG1α, but not PKG1β, immunoreactivity, was coincident with that of neuronal markers (HuC/D; β3‐tubulin) in both enteric plexuses. PKG1α immunoreactivity also co‐localized with the immunoreactivities of the IPAN markers, calbindin (100%; myenteric plexus) and cytoplasmic NeuN (98 ± 1% submucosal plexus). CGRP‐immunoreactive DRG neurons, identified as visceral afferents by retrograde transport, were PKG1α–immunoreactive. We used intraluminal cholera toxin to determine whether PKG1α was necessary to enable stimulation of the mucosa to activate Fos in enteric neurons. Tetrodotoxin (1.0 µM), low Ca²⁺/high Mg²⁺ media, and the PKG inhibitor, N46 (100 µM), all inhibited Fos activation in myenteric neurons. N46 also concentration dependently inhibited peristaltic reflexes in isolated preparations of distal colon (IC50 = 83.3 ± 1.3 µM).
Conclusions & Inferences
These data suggest that PKG1α is present and functionally important in IPANs and visceral afferent nociceptive neurons.
Interstitial cells of Cajal (ICC) generate pacemaker activity responsible for phasic contractions in colonic segmentation and peristalsis. ICC along the submucosal border (ICC-SM) contribute to mixing and more complex patterns of colonic motility. We show the complex patterns of Ca ²⁺ signaling in ICC-SM and the relationship between ICC-SM Ca ²⁺ transients and activation of SMCs using optogenetic tools. ICC-SM displayed rhythmic firing of Ca ²⁺ transients ~15 cpm and paced adjacent SMCs. The majority of spontaneous activity occurred in regular Ca ²⁺ transients clusters (CTCs) that propagated through the network. CTCs were organized and dependent upon Ca ²⁺ entry through voltage-dependent Ca ²⁺ conductances, L- and T-type Ca ²⁺ channels. Removal of Ca ²⁺ from the external solution abolished CTCs. Ca ²⁺ release mechanisms reduced the duration and amplitude of Ca ²⁺ transients but did not block CTCs. These data reveal how colonic pacemaker ICC-SM exhibit complex Ca ²⁺ firing patterns and drive smooth muscle activity and overall colonic contractions.
Key points
Platelet‐derived growth factor receptor‐α (PDGFRα) is a novel biomarker along with smooth myosin heavy chain for the pacemaker cells (previously termed ‘atypical’ smooth muscle cells) in the murine and cynomolgus monkey pelvis–kidney junction.
PDGFRα⁺ cells present in adventitial and urothelial layers of murine renal pelvis do not express smooth muscle myosin heavy chain (smMHC) but are in close apposition to nerve fibres.
Most c‐Kit⁺ cells in the renal pelvis are mast cells. Mast cells (CD117⁺/CD45⁺) are more abundant in the proximal renal pelvis and pelvis–kidney junction regions whereas c‐Kit⁺ interstitial cells (CD117⁺/CD45⁻) are found predominantly in the distal renal pelvis and ureteropelvic junction.
PDGFRα⁺ cells are distinct from c‐Kit⁺ interstitial cells.
A subset of PDGFRα⁺ cells express the Ca²⁺‐activated Cl⁻ channel, anoctamin‐1, across the entire renal pelvis.
Spontaneous Ca²⁺ transients were observed in c‐Kit⁺ interstitial cells, smMHC⁺ PDGFRα cells and smMHC⁻ PDGFRα cells using mice expressing genetically encoded Ca²⁺ sensors.
Abstract
Rhythmic contractions of the renal pelvis transport urine from the kidneys into the ureter. Specialized pacemaker cells, termed atypical smooth muscle cells (ASMCs), are thought to drive the peristaltic contractions of typical smooth muscle cells (TSMCs) in the renal pelvis. Interstitial cells (ICs) in close proximity to ASMCs and TSMCs have been described, but the role of these cells is poorly understood. The presence and distributions of platelet‐derived growth factor receptor‐α⁺ (PDGFRα⁺) ICs in the pelvis–kidney junction (PKJ) and distal renal pelvis were evaluated. We found PDGFRα⁺ ICs in the adventitial layers of the pelvis, the muscle layer of the PKJ and the adventitia of the distal pelvis. PDGFRα⁺ ICs were distinct from c‐Kit⁺ ICs in the renal pelvis. c‐Kit⁺ ICs are a minor population of ICs in murine renal pelvis. The majority of c‐Kit⁺ cells were mast cells. PDGFRα⁺ cells in the PKJ co‐expressed smooth muscle myosin heavy chain (smMHC) and several other smooth muscle gene transcripts, indicating these cells are ASMCs, and PDGFRα is a novel biomarker for ASMCs. PDGFRα⁺ cells also express Ano1, which encodes a Ca²⁺‐activated Cl⁻ conductance that serves as a primary pacemaker conductance in ICs of the GI tract. Spontaneous Ca²⁺ transients were observed in c‐Kit⁺ ICs, smMHC⁺ PDGFRα cells and smMHC⁻ PDGFRα cells using genetically encoded Ca²⁺ sensors. A reporter strain of mice with enhanced green fluorescent protein driven by the endogenous promotor for Pdgfra was shown to be a powerful new tool for isolating and characterizing the phenotype and functions of these cells in the renal pelvis.
Key points
Rhythmic action potentials and intercellular Ca²⁺ waves are generated in smooth muscle cells of colonic longitudinal muscles (LSMC).
Longitudinal muscle excitability is tuned by input from subserosal ICC (ICC‐SS), a population of ICC with previously unknown function.
ICC‐SS express Ano1 channels and generate spontaneous Ca²⁺ transients in a stochastic manner.
Release of Ca²⁺ and activation of Ano1 channels causes depolarization of ICC‐SS and LSMC, leading to activation of L‐type Ca²⁺ channels, action potentials, intercellular Ca²⁺ waves and contractions in LSMC.
Nitrergic neural inputs regulate the Ca²⁺ events in ICC‐SS.
Pacemaker activity in longitudinal muscle is an emergent property as a result of integrated processes in ICC‐SS and LSMC.
Abstract
Much is known about myogenic mechanisms in circular muscle (CM) in the gastrointestinal tract, although less is known about longitudinal muscle (LM). Two Ca²⁺ signalling behaviours occur in LM: localized intracellular waves not causing contractions and intercellular waves leading to excitation‐contraction coupling. An Ano1 channel antagonist inhibited intercellular Ca²⁺ waves and LM contractions. Ano1 channels are expressed by interstitial cells of Cajal (ICC) but not by smooth muscle cells (SMCs). We investigated Ca²⁺ signalling in a novel population of ICC that lies along the subserosal surface of LM (ICC‐SS) in mice expressing GCaMP6f in ICC. ICC‐SS fired stochastic localized Ca²⁺ transients. Such events have been linked to activation of Ano1 channels in ICC. Ca²⁺ transients in ICC‐SS occurred by release from stores most probably via inositol trisphosphate receptors. This activity relied on influx via store‐operated Ca²⁺ entry and Orai channels. No voltage‐dependent mechanism that synchronized Ca²⁺ transients in a single cell or between cells was found. Nitrergic agonists inhibited Ca²⁺ transients in ICC‐SS, and stimulation of intrinsic nerves activated nitrergic responses in ICC‐SS. Cessation of stimulation resulted in significant enhancement of Ca²⁺ transients compared to the pre‐stimulus activity. No evidence of innervation by excitatory, cholinergic motor neurons was found. Our data suggest that ICC‐SS contribute to regulation of LM motor activity. Spontaneous Ca²⁺ transients activate Ano1 channels in ICC‐SS. Resulting depolarization conducts to SMCs, depolarizing membrane potential, activating L‐type Ca²⁺ channels and initiating contraction. Rhythmic electrical and mechanical behaviours of LM are an emergent property of SMCs and ICC‐SS.
Key points:
Enteric neurotransmission is essential for gastrointestinal (GI) motility, although the cells and conductances responsible for post-junctional responses are controversial. The calcium-activated chloride conductance (CaCC), anoctamin-1 (Ano1), was expressed by intramuscular interstitial cells of Cajal (ICC-IM) in proximal stomach and not resolved in smooth muscle cells (SMCs). Cholinergic nerve fibres were closely apposed to ICC-IM. Conductances activated by cholinergic stimulation in isolated ICC-IM and SMCs were determined. A CaCC was activated by carbachol in ICC-IM and a non-selective cation conductance in SMCs. Responses to cholinergic nerve stimulation were studied. Excitatory junction potentials (EJPs) and mechanical responses were evoked in wild-type mice but absent or greatly reduced with knockout/down of Ano1. Drugs that block Ano1 inhibited the conductance activated by carbachol in ICC-IM and EJPs and mechanical responses in tissues. The data of the present study suggest that electrical and mechanical responses to cholinergic nerve stimulation are mediated by Ano1 expressed in ICC-IM and not SMCs.
Abstract:
Enteric motor neurotransmission is essential for normal gastrointestinal (GI) motility. Controversy exists regarding the cells and ionic conductance(s) that mediate post-junctional neuroeffector responses to motor neurotransmitters. Isolated intramuscular ICC (ICC-IM) and smooth muscle cells (SMCs) from murine fundus muscles were used to determine the conductances activated by carbachol (CCh) in each cell type. The calcium-activated chloride conductance (CaCC), anoctamin-1 (Ano1) is expressed by ICC-IM but not resolved in SMCs, and CCh activated a Cl-conductance in ICC-IM and a non-selective cation conductance in SMCs. We also studied responses to nerve stimulation using electrical-field stimulation (EFS) of intact fundus muscles from wild-type and Ano1 knockout mice. EFS activated excitatory junction potentials (EJPs) in wild-type mice, although EJPs were absent in mice with congenital deactivation of Ano1 and greatly reduced in animals in which the CaCC-Ano1 was knocked down using Cre/loxP technology. Contractions to cholinergic nerve stimulation were also greatly reduced in Ano1 knockouts. SMCs cells also have receptors and ion channels activated by muscarinic agonists. Blocking acetylcholine esterase with neostigmine revealed a slow depolarization that developed after EJPs in wild-type mice. This depolarization was still apparent in mice with genetic deactivation of Ano1. Pharmacological blockers of Ano1 also inhibited EJPs and contractile responses to muscarinic stimulation in fundus muscles. The data of the present study are consistent with the hypothesis that ACh released from motor nerves binds muscarinic receptors on ICC-IM with preference and activates Ano1. If metabolism of acetylcholine is inhibited, ACh overflows and binds to extrajunctional receptors on SMCs, eliciting a slower depolarization response.
The anatomic orientation of the gastric smooth muscle layers (longitudinal, circular, and oblique) serves the need to regulate accommodation, dispersion, trituration, and emptying of food. Gastric smooth muscle is innervated by extrinsic nerves that relay information to and from extrinsic ganglia, spinal cord, and central nervous system, and by intrinsic nerves in the gastric wall. This chapter summarizes neurons that control digestive functions, their predominant transmitters and stimulatory or inhibitory actions. The small intestine modulates gastric emptying and plays a pivotal role in regulating gastric and pyloric motor activity. Interruption of vagal innervation of the proximal stomach does not modify emptying of indigestible spheres, whereas distal gastric vagotomy delays their passage into the duodenum. Feedback inhibition of emptying by intestinal factors is crucial to regulating duodenal caloric delivery. The effects of many hormones and neurotransmitters on gastric emptying reflect their effects on gastric motility.
Irritable bowel syndrome (IBS) is a functional bowel disease with a complicated etiopathogenesis, often characterized by gastrointestinal motility disorder and high visceral sensitivity. IBS is a comprehensive multi-systemic disorder, with the interaction of multiple factors, such as mental stress, intestinal function and flora, heredity, resulting in the disease. The existence of a common mechanism underlying the aforementioned factors is currently unknown. The lack of therapies that comprehensively address the disease symptoms, including abdominal pain and diarrhea, is a limitation of current IBS management. The current review has explored the role of the SCF/c-Kit receptor/ligand system in IBS. The SCF/c-Kit system constitutes a classical ligand/receptor tyrosine kinase signaling system that mediates inflammation and smooth muscle contraction. Additionally, it provides trophic support to neural crest-derived cell types, including the enteric nervous system and mast cells. The regulation of SCF/c-Kit on the interstitial cells of Cajal (ICC) suggest that it may play a key role in the aberrant intestinal dynamics and high visceral sensitivity observed in IBS. The role of the SCF/c-Kit system in intestinal motility, inflammation and nerve growth has been reported. From the available biomedical evidence on the pathogenesis of IBS, it has been concluded that the SCF-c-Kit system is a potential therapeutic target for rational drug design in the treatment of IBS.
In order for the gut to perform essential functions, including moving contents along its length via the organised movement of peristalsis, as well as absorption of water and electrolytes, secretion from glands and regulation of blood flow, the integrated function of multiple tissues and cell types must occur. The mature, functioning neuromuscular system of the gut is comprised of smooth muscle cells, neurons and glial cells of the enteric nervous system (ENS) and interstitial cells of Cajal (ICCs). These diverse components arise from distinct sources during development and must, during the course of embryogenesis, acquire appropriate integration to enable a functioning neuromuscular system to commence coordinated activity around birth. Here, we utilise information gleaned from studies in animal models such as mouse, chick, guinea pig, and zebrafish, as well as human studies, to describe the development of each constituent part of the neuromuscular system as well as to outline how these component parts become integrated into a functioning whole. Moreover, our discussions touch on diseases affecting development of the enteric neuromuscular system, notably Hirschsprung’s disease (HSCR), one of the most common gut motility disorders.
Objectives:
To study the c-Kit expression in the gallbladder of cholesterol lithogenic guinea pig model and the effect of Artemisia capillaris Thunb on interstitial cells of Cajal (ICCs).
Materials and methods:
A total of 45 guinea pigs were randomly assigned into three groups: the control group (guinea pigs fed a standard diet, normal group); the model group (guinea pigs fed a cholesterol gallstone-inducing diet); and the Chinese medicine group (guinea pigs fed the cholesterol gallstone-inducing diet and treated with A. capillaris through intragastric administration, therapy group). Each group had 15 guinea pigs. The gallbladders of the guinea pigs were harvested after 8 weeks. C-Kit expression was detected using an immunohistochemistry staining, real-time PCR, and Western blot analyses. The effect of A. capillaris on ICCs was evaluated by muscle strip contraction experiments.
Results:
C-Kit expression significantly decreased in the gallbladder of model group, but increased in the Chinese medicine group. The Contractility of guinea pig gallbladder muscle strip significantly improved in the Chinese medicine group.
Conclusion:
Our results indicated that A. capillaris improves gallbladder impairment by up-regulating c-Kit expression, and it also can improve the contractile response of in vitro guinea pig gallbladder muscle strips.
Interstitial cells of mesenchymal origin form gap junctions with smooth muscle cells in visceral smooth muscles and provide important regulatory functions. In gastrointestinal (GI) muscles, there are two distinct classes of interstitial cells, c-Kit+ interstitial cells of Cajal and PDGFRα+ cells, that regulate motility patterns. Loss of these cells may contribute to symptoms in GI motility. disorders.
The colonic migrating motor complex (CMMC) is a critical neurally mediated, cyclical contractile and electrical event. CMMC is the primary motor pattern underlying fecal pellet propulsion along the murine colon. Abnormal CMMC has important implications in a number of gastrointestinal disorders, especially slow transit constipation. This review focuses on the mechanisms involved in producing and propagating the CMMC, which is likely dependent on mucosal and neuronal serotonin and pacemaker interstitial cells of Cajal networks and how peristaltic reflexes or occult reflexes affect them, and emphasizes the important role of intrinsic primary afferent neurons, ascending excitatory and descending inhibitory neural pathways. In addition to these, we also introduce some new tools to detect specific neuronal activity so as to offer some exciting insights into the role of 5-hydroxytryptamine in colonic motility.
In order for the gut to perform essential functions, including moving contents along its length via the organized movement of peristalsis, as well as absorption of water and electrolytes, secretion from glands, and regulation of blood flow, the integrated function of multiple tissues and cell types must occur. The mature, functioning neuromuscular system of the gut is composed of smooth muscle cells, neurons and glial cells of the enteric nervous system (ENS), and interstitial cells of Cajal (ICC). These diverse components arise from distinct sources during development, and must, during the course of embryogenesis, acquire appropriate integration to enable a functioning neuromuscular system to commence coordinated activity around birth. Here, we utilize information gleaned from studies in animal models such as mouse, chick, guinea pig, and zebrafish, as well as human studies, to describe the development of each constituent part of the neuromuscular system as well as to outline how these component parts become integrated into a functioning whole. Moreover, our discussions touch on diseases affecting development of the enteric neuromuscular system, notably Hirschsprung’s disease (HSCR), one of the most common gut motility disorders.
Coordinated movements of the gastrointestinal tract are regulated by multiple mechanisms including intrinsic and extrinsic neurons, interstitial cells of Cajal (ICC), and myogenic mechanisms. Studies using laboratory animals have shown that although enteric neurons develop early, the first gastrointestinal motility patterns are myogenic, and not neurally mediated. However, neurally mediated contractile activity is prominent by birth, and is essential for propulsive activity as shown by the bowel obstruction that occurs proximal to the aganglionic region in infants with Hirschsprung’s disease. The development of ICC requires signaling via the tyrosine kinase receptor, Kit. Genetic alterations of Kit, and reduced ICC density, have recently been linked to a severe case of idiopathic constipation and megacolon in a child. Studies in preterm and term humans have shown that esophageal peristalsis and sphincter function mature during the late fetal and early postnatal stages. Little is known about the development of motility in the small and large bowel of human infants.
Lactobacillus plantarum, as a probiotic, has many functional properties in human intestinal tract. This study examined the effects of L. plantarum NCU116 on loperamide-induced constipation in a mouse model. Loperamide (5 mg kg(-1)) was injected subcutaneously to induce constipation. Animals were divided to five groups: normal group, constipation group, constipation plus three doses of L. plantarum NCU116 groups (NCU116-L, 10(7) CFU/mL; NCU116-M, 10(8) CFU/mL; NCU116-H, 10(9) CFU/mL; respectively). Mice were treated with the probiotic for 15 d to assess the anti-constipation effects. Fecal parameters, intestinal transit ratio and the production of fecal short chain fatty acids, histological of colon and immunohistochemical in colonic interstitial cells of Cajal (ICC) by c-kit were all improved in L. plantarum NCU116-treated mice as compared to the constipation group. These results demonstrate that L. plantarum NCU116 enhanced gastrointestinal transit and alleviated in mice with loperamide-induced constipation.
Smooth muscles are complex tissues containing a variety of cells in addition to muscle cells. Interstitial cells of mesenchymal origin interact with and form electrical connectivity with smooth muscle cells in many organs, and these cells provide important regulatory functions. For example, in the gastrointestinal tract, interstitial cells of Cajal (ICC) and PDGFRα(+) cells have been described, in detail, and represent distinct classes of cells with unique ultrastructure, molecular phenotypes, and functions. Smooth muscle cells are electrically coupled to ICC and PDGFRα(+) cells, forming an integrated unit called the SIP syncytium. SIP cells express a variety of receptors and ion channels, and conductance changes in any type of SIP cell affect the excitability and responses of the syncytium. SIP cells are known to provide pacemaker activity, propagation pathways for slow waves, transduction of inputs from motor neurons, and mechanosensitivity. Loss of interstitial cells has been associated with motor disorders of the gut. Interstitial cells are also found in a variety of other smooth muscles; however, in most cases, the physiological and pathophysiological roles for these cells have not been clearly defined. This review describes structural, functional, and molecular features of interstitial cells and discusses their contributions in determining the behaviors of smooth muscle tissues.
Smooth muscle layers of the gastrointestinal tract consist of a heterogeneous population of cells that include enteric neurons, several classes of interstitial cells of mesenchymal origin, a variety of immune cells and smooth muscle cells (SMCs). Over the last number of years the complexity of the interactions between these cell types has begun to emerge. For example, interstitial cells, consisting of both interstitial cells of Cajal (ICC) and platelet-derived growth factor receptor alpha (PDGFRα+) cells generate pacemaker activity throughout the gastrointestinal (GI) tract and also transduce enteric motor nerve signals and mechanosensitivity to adjacent SMCs. ICC and PDGFRα+ cells are electrically coupled to SMCs possibly via gap junctions forming a multicellular functional syncytium termed the SIP syncytium. Cells that make up the SIP syncytium are highly specialized containing unique receptors, ion channels and intracellular signaling pathways that regulate the excitability of GI muscles. The unique role of these cells in coordinating GI motility is evident by the altered motility patterns in animal models where interstitial cell networks are disrupted. Although considerable advances have been made in recent years on our understanding of the roles of these cells within the SIP syncytium, the full physiological functions of these cells and the consequences of their disruption in GI muscles have not been clearly defined. This review gives a synopsis of the history of interstitial cell discovery and highlights recent advances in structural, molecular expression and functional roles of these cells in the GI tract.
Previous studies have shown that cGMP-dependent protein kinase (PKG) act on several targets in the contractile pathway to reduce intracellular Ca2+ and/or augment RhoA-regulated myosin light chain phosphatase (MLCP) activity and cause muscle relaxation. Recent studies have identified a novel protein M-RIP that associates with MYPT1, the regulatory subunit of MLCP. Herein, we examine whether PKG enhance MLCP activity downstream of Ca2+ and RhoA via phosphorylation of M-RIP in gastric smooth muscle cells. Treatment of permeabilized muscle cells with 10 μM Ca2+ caused an increase in MLC20 phosphorylation and muscle contraction, but had no effect on Rho kinase activity. Activators of PKG (GSNO or cGMP) decreased MLC20 phosphorylation and contraction in response to 10 μM Ca2+, implying existence of inhibitory mechanism independent of Ca2+ and RhoA. The effect of PKG on Ca2+-induced MLC20 phosphorylation was attenuated by M-RIP siRNA. Both GSNO and 8-pCPT-cGMP induced phosphorylation of M-RIP; phosphorylation was accompanied by an increase in the association of M-RIP with MYPT1 and MLCP activity. Taken together, these results provide evidence that PKG induces phosphorylation of M-RIP and enhances its association with MYPT1 to augment MLCP activity and MLC20 dephosphorylation and inhibits muscle contraction, downstream of Ca2+- or RhoA-dependent pathways.
Interstitial cells of Cajal (ICC) have been suggested as pacemaker cells in the gastrointestinal tract. A method was developed to isolate ICC from the slow-wave pacemaker region of the canine proximal colon. These cells were identified under phase-contrast microscopy, and their identity was verified by comparing their ultrastructure with the morphology of ICC in situ. Patch-clamp experiments demonstrated that these cells are excitable; voltage-dependent inward and outward currents were elicited by depolarization. Inward current transients were identified as calcium currents. A portion of the outward current appears to be due to Ca2+-activated K channels commonly expressed in these cells. ICC were also spontaneously active, generating electrical depolarizations similar in waveform to slow-wave events of intact colonic muscles. These findings are consistent with the hypothesis that ICC initiate rhythmicity in the colon.
We have studied the layers of the muscular coat of the guinea-pig small intestine after enzymatic and chemical removal of extracellular connective tissue. The cells of the longitudinal muscle layer are wider, have rougher surfaces, more finger-like processes and more complex terminations, but fewer intercellular junctions than cells in the circular muscle layer. A special layer of wide, flat cells with a dense innervation exists at the inner margin of the circular muscle layer, facing the submucosa. The ganglia of the myenteric and submucosal plexuses are covered by a smooth basal lamina, a delicate feltwork of collagen fibrils, and innumerable connective tissue cells. The neuronal and glial cell processes at the surface of ganglia form an interlocking mosaic, which is loosely packed in newborn and young animals, but becomes tightly packed in adults. The arrangement of glial cells becomes progressively looser along finer nerve bundles. Single varicose nerve fibres are rarely exposed, but multiaxonal bundles are common. Fibroblast-like cells of characteristic shape and orientation are found in the serosa; around nerve ganglia; in the intermuscular connective tissue layer and in the circular muscle, where they bridge nerve bundles and muscle cells; at the submucosal face of the special, flattened inner circular muscle layer; and in the submucosa. Some of these fibroblast-like cells correspond to interstitial cells of Cajal. Other structures readily visualized by scanning electron microscopy are blood and lymphatic vessels and their periendothelial cells. The relationship of cellular elements to connective tissue was studied with three different preparative procedures: (1) freeze-cracked specimens of intact, undigested intestine; (2) 'stretch preparations' of longitudinal muscle with adhering myenteric plexus; (3) sheets of submucosal collagen bundles from which all cellular elements had been removed by prolonged detergent extraction.
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.
The pacemaker activity in the mammalian gut is responsible for generating anally propagating phasic contractions. The cellular basis for this intrinsic activity is unknown. The smooth muscle cells of the external muscle layers and the innervated cellular network of interstitial cells of Cajal, which is closely associated with the external muscle layers of the mammalian gut, have both been proposed to stimulate pacemaker activity. The interstitial cells of Cajal were identified in the last century but their developmental origin and function have remained unclear. Here we show that the interstitial cells of Cajal express the Kit receptor tyrosine kinase. Furthermore, mice with mutations in the dominant white spotting (W) locus, which have cellular defects in haematopoiesis, melanogenesis and gametogenesis as a result of mutations in the Kit gene, also lack the network of interstitial cells of Cajal associated with Auerbach's nerve plexus and intestinal pacemaker activity.
A subpopulation of interstitial cells (ICs) are interposed between nerve terminals and smooth muscle cells in the gastrointestinal tract and may participate in neuromuscular transmission. These cells appear to be targets for NO released from enteric inhibitory nerves and respond to exogenous NO with: (i) an elevation in cGMP levels; (ii) an increase in intracellular Ca2+; (iii) and release of a diffusible substance that has tentatively been identified as NO. For the latter to be possible, ICs must express a constitutive isoform of NOS. This study characterized the expression of NOS-like immunoreactivity (NOS-LI) in ICs of the canine colon using 3 antibodies raised against the 2 known constitutive forms of NOS (i.e., neural (nNOS) and endothelial (eNOS) isoforms). Antibodies raised against eNOS and an antibody raised against rat cerebellar nNOS labeled ICs along the submucosal surface of the circular muscle layer (IC-SM), along the surface of septa that separate the circular muscle into fiber bundles (IC-SM), and in the myenteric region between the circular and longitudinal muscle layers (IC-MY). Another antibody raised against rat cerebellar nNOS failed to label ICs. Cultured IC-SM also expressed NOS-LI, suggesting that this feature of the IC phenotype survives culture conditions. Arteriolar endothelial cells in the canine colon were labeled with the same 2 antibodies that labeled ICs, suggesting there are significant structural similarities between NO synthases in ICs and endothelial cells. The data suggest that IC-SM and IC-MY express a constitutive form of NOS. Synthesis of NO by ICs may influence electrical rhythmicity and may serve to amplify and even propagate enteric inhibitory neurotransmission.
Studies of the enteric nervous system have proved to be important in the development of new concepts of the chemical nature of transmission from neurons. In particular, they have revealed the multiplicity of influences that peptides can have on transmission, such as their action as primary transmitters, and the fact that they often act as co-transmitters in enteric neurons. However, in other cases no roles can be attributed to neuropeptides in enteric neurons, and their involvement in short-term changes in excitability seems minor.
Slow waves in the small intestine seem to arise in plexuses of neurites with interstitial cells of Cajal. In the colon, slow waves appear to arise at the circular muscle - submucosal interface. We therefore sought a plexus at this surface in the colon in the cat, dog, ferret, opossum, rabbit, rat, guinea-pig and man. Segments from all levels of the colon were stained by the Champy-Maillet osmic acid-zinc iodide method and cut into serial 25 micron sections in the plane of the muscle layers. A dense network of neurites with abundant interstitial cells of Cajal was found at the circular muscle - submucosal interface in all species except rabbit. Neurites in this plexus appeared to arise from the deep plexus of the submucosa (Schabadasch's or Henle's plexus). It was not found in the small intestine and stomach. A similar plexus was found in the interstices of the myenteric plexus in the colon. Interstitial cells of Cajal in both plexuses were positive for the NADH-diaphorase stain, but not for silver impregnation. The possible roles of the plexuses of neurites and interstitial cells of Cajal at the circular muscle - submucosal interface and at the plane of the myenteric plexus in the generation of rhythmic activity in the colon are discussed.
There is experimental evidence suggesting that the interstitial cells of Cajal are essential for rhythmic slow waves of the smooth muscle layers of the mammalian small intestine. Different investigators have identified them variously as modified neurons, glia, fibroblasts or modified smooth muscle cells. Since histological categorization bears on understanding their function, we have examined the immunoreactivity of the myenteric plexus of the rat small intestine, paying special attention to the cell type identified as Thuneberg's Type I-ICC. Polyclonal and monoclonal antisera directed against 4 intermediate filament proteins: neurofilament protein, glial fibrillary acidic protein, vimentin and desmin were used. In addition, antisera directed against neuron-specific enolase, substance P and vasoactive intestinal polypeptide were also tested for reactivity. Type I-ICCs were immunonegative to all the antisera directed against intermediate filament proteins and neuropeptides. However, some Type I-ICCs were immunopositive to antisera against neuron-specific enolase. On the basis of these results and the distribution of immunoreactivities to these kinds of antisera in other tissues, we suggest that Type I-ICCs are distinct from typical myenteric neurons, from glia, from fibroblasts and from smooth muscle fibers. Staining with antiserum against neuron-specific enolase suggests a relation to some type of neuron.
In vivo injection of a neutralizing, monoclonal antibody (ACK2) to the receptor tyrosine kinase (c-kit) disrupts the normal motility patterns of the mouse small intestine. Immunohistochemical studies showed that cells expressing c-kit-like immunoreactivity (c-kit-LI) decreased in numbers in response to ACK2, but the identity of these cells is unknown. We investigated the identity and development of the cells that express c-kit-LI in the mouse small intestine and colon. Cells in the region of the myenteric plexus and deep muscular plexus of the small intestine and in the subserosa, in the myenteric plexus region, within the circular and longitudinal muscle layers, and along the submucosal surface of the circular muscle in the colon were labeled with ACK2. The distribution of cells that express c-kit-LI was the same as that of interstitial cells (ICs). In whole-mount preparations cells with c-kit-LI were interconnected, forming a network similar to the network formed by cells that stained with methylene blue, which has been used as a marker for ICs in the mouse gastrointestinal tract. Immunocytochemistry verified that ICs were labeled with ACK2. Multiple injections of animals with ACK2 between days 0 and 8 post partum (pp) caused a dramatic reduction in the number of ICs compared to control animals. From an ultrastructural point of view, the proliferation and development appeared to be suppressed in some classes of ICs, while others displayed an altered course of development. Functional studies showed that the decrease in ICs was accompanied by a loss of electrical rhythmicity in the small intestine and reduced neural responses in the small bowel and colon. Morphological experiments showed that c-kit-positive cells are ICs, and physiological evidence reinforced the concept that ICs are involved in generation of rhythmicity and translation of neural inputs in gastrointestinal smooth muscles. Controlling the development of ICs provides a powerful new tool for the investigation of the physiological role of these cells.
This study has demonstrated that cells stained using the zinc iodide-osmic acid (ZIO) method have the same fine structural features as those of fibroblasts. They have a well developed Golgi apparatus, granular endoplasmic reticulum and many mitochondria. They have no basal lamina. They are distributed in association with the deep muscular plexus, within the outer circular muscle layer and in the space between the circular and longitudinal muscle layers. Since this staining method is believed to co-stain nerves and interstitial cells of Cajal, we concluded that these ZIO-positive, fibroblast-like cells represent at least some, if not all, of the interstitial cells which appeared in the original description by Cajal.
In gastrointestinal muscles special cells, referred to an interstitial cells, may be involved in pacemaking and transduction of inputs from the enteric nervous system. We have used a modification of the NADH diaphorase method to characterize the distribution of interstitial cells in the muscularis externa of the canine colon. The staining product of the NADH diaphorase reaction is useful because it allows light and electron microscopic studies to be performed with the same marker. Therefore rigorous identification of the cells observed at the light microscopic level could be made by electron microscopy. We were able to label at least three classes of interstitial cells: (1) at the submucosal surface of the circular muscle layer; (2) within the thickness of the circular and longitudinal muscle layers; and (3) in the region of the myenteric plexus. This technique also labeled cell bodies and initial segments of processes of Dogiel type II neurones in enteric ganglia. Nerve fibres within the muscle layers did not exhibit NADH diaphorase activity. This study has identified the interstitial cells within the circular and longitudinal muscle layers and shows the arrangement of these cells in a three-dimensional network.
1. Interstitial cells of Cajal (ICs) have been proposed as pacemakers in the gastrointestinal tract. We studied the characteristics and distribution of ICs and electrical activity of small intestinal muscles from mice with mutations at the dominant-white spotting/c-kit (W) locus because the tyrosine kinase function of c-kit may be important in the development of the IC network. 2. W/WV mutants (days 3-30 postpartum) had few ICs in the myenteric plexus region compared with wild type (+/+) siblings. The few ICs present were associated with neural elements and lay between myenteric ganglia and the longitudinal muscle layer. 3. Electrical recordings from intestinal muscle strips showed that electrical slow waves were always present in muscles of +/+ siblings, but were absent in W/WV mice. 4. Muscles from W/WV mice responded to stimulation of intrinsic nerves. Neural responses, attributed to the release of acetylcholine, nitric oxide and other unidentified transmitters, were recorded. 5. These findings are consistent with the hypothesis that ICs are a critical element in the generation of electrical rhythmicity in intestinal muscles. The data also show that neural regulation of gastrointestinal muscles can develop independently of the IC network. 6. W locus mutants provide a powerful new model for studies of the physiological role of ICs and the significance of electrical rhythmicity to normal gastrointestinal motility.
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.
Requirement of c-kit for de-velopment of intestinal pacemaker system Immunocytochemistry of the interstitial cells of Cajal in the rat intestine
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Nitric oxide targets in the guinea-pig intestine identified by induc-tion of cyclic GMP immunoreactivity Ultrastructure of the zinc iodide-osmic acid stained cells in the guinea pig small intestine
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Histologi-cal identification of the interstitial cells of Cajal in the guinea-pig small intestine
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Identifi-cation of interstitial cells in canine proximal Colon using NAOH diaphorase histochemistry
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Impaired development of interstitial cells and intestinal elec-trical rhythmicity in steel mutants Use of rhodamine-123 to label and lesion interstitial cells of Cajal in canine colonic circular muscle
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