Pivotal role of the interstitial cells of Cajal in the nitric oxide signaling pathway of rat small intestine. Morphological evidence

The nitric oxide (NO) signaling pathway is a major nonadrenergic-noncholinergic transmitter mechanism in the enteric nervous system. Our aim was to localize the enzymes in question, i.e., neuronal nitric oxide synthase (nNOS), soluble guanylate cyclase (sGC), and cGMP-dependent kinase type I (cGK-I) in rat small intestine by indirect immunofluorescence. nNOS staining was found in neurons of the myenteric plexus and in varicose nerve fibers mainly in the circular muscle layer. The cells positive for neurokinin-1 (NK-1) receptor and c-kit (interstitial cells of Cajal, ICC) in the deep muscular plexus (DMP) did not show nNOS reactivity, but nNOS-positive nerve fibers were directly adjacent to them. sGC was found in flattened cells surrounding myenteric ganglia (periganglionic cells, PGC), in ICC of the DMP, faintly in smooth muscle cells (SMC), and in cells perivascularly scattered throughout the circular muscle layer. cGK-I immunoreactivity was found abundantly in PGC (which presumably are ICC), in ICC of DMP, in SMC of the innermost circular and longitudinal muscle layers, but less intensively in the outer circular layer. Weak cGK-I staining occurred in nerve cells within the myenteric and submucosal plexus. Conclusively the key enzymes of the NO signaling pathway are differentially distributed: Occurrence of nNOS exclusively in neurons and the presence of sGC and cGK-I predominantly in ICC suggest a sequence of neuronal NO release, activation of ICC, and consecutive smooth muscle relaxation. ICC of the DMP seem to be the primary targets for neurally released NO.     

Nitric oxide synthase immunoreactivity in the enteric nervous system of the developing human digestive tract

We have investigated indirectly the presence of nitric oxide in the enteric nervous system of the digestive tract of human fetuses and newborns by nitric oxide synthase (NOS) immunocytochemistry and nicotinamide adenine dinucleotide phosphate diaphorase (NADPHd) histochemistry. In the stomach, NOS immunoactivity was confined to the myenteric plexus and nerve fibres in the outer smooth musculature; few immunoreactive nerve cell bodies were found in ganglia of the outer submucous plexus. In the pyloric region, a few nitrergic perikarya were seen in the inner submucous plexus and some immunoreactive fibres were found in the muscularis mucosae. In the small intestine, nitrergic neurons clustered just underneath or above the topographical plane formed by the primary nerve strands of the myenteric plexus up to the 26th week of gestation, after which stage, they occurred throughout the ganglia. Many of their processes contributed to the dense fine-meshed tertiary nerve network of the myenteric plexus and the circular smooth muscle layer. NOS-immunoreactive fibres directed to the circular smooth muscle layer originated from a few NOS-containing perikarya located in the outer submucous plexus. In the colon, caecum and rectum, labelled nerve cells and fibres were numerous in the myenteric plexus; they were also found in the outer submucous plexus. The circular muscle layer had a much denser NOS-immunoreactive innervation than the longitudinally oriented taenia. The marked morphological differences observed between nitrergic neurons within the developing human gastrointestinal tract, together with the typical innervation pattern in the ganglionic and aganglionic nerve networks, support the existenc of distinct subpopulations of NOS-containing enterice neurons acting as interneurons or (inhibitory) motor neurons.     

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

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

Projections of neurochemically specified neurons in the porcine colon

The intramural projections of nerve cells containing serotonin (5-HT), calcitonin gene-related peptide (CGRP), vasoactive intestinal peptide (VIP) and nitric oxide synthase or reduced nicotinamide adenine dinucleotide phosphate diaphorase (NOS/NADPHd) were studied in the ascending colon of 5- to 6-week-old pigs by means of immunocytochemistry and histochemistry in combination with myectomy experiments. In control tissue of untreated animals, positive nerve cells and fibres were common in the myenteric and outer submucous plexus and, except for 5-HT-positive perikarya, immunoreactive cell bodies and fibres were also observed in the inner submucous plexus. VIP- and NOS/NADPHd-positive nerve fibres occurred in the ciruclar muscle layer while VIP was also abundant in nerve fibres of the mucosal layer. 5-HT- and CGRP-positive nerve fibres were virtually absent from the aganglionic nerve networks. In the submucosal layer, numerous paravascular CGRP-immunoreactive (IR) nerve fibres were encountered. Myectomy studies revealed that 5-HT-, CGRP-, VIP- and NOS/NADPHd-positive myenteric neurons all displayed anal projections within the myenteric plexus. In addition, some of the serotonergic myenteric neurons projected anally to the outer submucous plexus, whereas a great number of the VIP-ergic and nitrergic myenteric neurons send their axons towards the circular muscle layer. The possible function of these nerve cells in descending nerve pathways in the porcine colon is discussed in relation to the distribution pattern of their perikarya and processes and some of their morphological characteristics.     

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

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

Three-dimensional structures of c-Kit-positive cellular networks in the guinea pig small intestine and colon

Cryosections and whole-mount preparations of the guinea pig small intestine and colon were single or double immunolabeled using the anti-c-Kit and protein gene product 9.5 antibodies. Immunolabeled specimens were observed under a confocal laser scanning microscope. The main findings of the present study are: (1) the distribution and profiles of three-dimensional structures of c-Kit-positive cellular networks in the small intestine and colon, and (2) the anatomical relations of c-Kit-positive cells to the enteric nerves in the layers. In the small intestine, c-Kit-positive cellular networks were observed at levels of the deep muscular plexus and myenteric plexus. The c-Kit-positive cellular networks ran along or overlay the nerve fibers at the deep muscular plexus, while they showed the reticular structures intermingled with the nerve elements at the myenteric plexus. In the colon, c-Kit-positive cellular networks were observed at levels of the submuscular plexus and myenteric plexus, and were further identified within the circular and longitudinal muscle layers as well as in the subserosal layer. In the circular muscle layer, c-Kit-positive cells surrounded the associated nerve fibers and extended several long processes toward the adjacent c-Kit-positive cells. The c-Kit-positive cellular networks within the longitudinal muscle layer as well as in the subserosal layer were not associated with the nerve fibers. In the layers of the intestinal wall with c-Kit-positive cells, the cellular networks of the interstitial cells were identified in ultrastructure. The characteristic profiles of c-Kit-positive cellular networks provide a morphological basis upon which to investigate the mechanisms regulating intestinal movement. Received: 14 July 1998 / Accepted: 2 September 1998     

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

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

3-D illustration of network orientations of interstitial cells of Cajal subgroups in human colon as revealed by deep-tissue imaging with optical clearing

Morphological changes of interstitial cells of Cajal (ICC) have been proposed to characterize motility disorders. However, a global view of the network orientations of ICC subgroups has not been established to illustrate their three-dimensional (3-D) architectures in the human colon. In this research, we integrate c-kit immunostaining, 3-D microscopy with optical clearing, and image rendering to present the location-dependent network orientations with high definition. Full-depth colonic tissues were obtained from colectomies performed for nonobstructing carcinoma. Specimens of colon wall were prepared away from the tumor site. C-kit and nuclear fluorescent staining were used to identify the ICC processes and cell body. Optical clearing was used to generate transparent colon specimens, which led to panoramic visualization of the fluorescence-labeled ICC networks at the myenteric plexus (ICC-MY), longitudinal (ICC-LM) and circular (ICC-CM) muscles, and submucosal boundary (ICC-SM) up to 300 μm in depth via confocal microscopy with subcellular level resolution. We observed four distinct network patterns: 1) periganglionic ICC-MY that connect with ICC-LM and ICC-CM, 2) plexuses of ICC-LM within the longitudinal muscle and extending toward the serosa, 3) repetitive and organized ICC-CM layers running parallel to the circular muscle axis and extending toward the submucosa, and 4) a condensed ICC-SM layer lining the submucosal border. Among the four patterns, the orderly aligned ICC-CM layers provide an appropriate target for quantitation. Our results demonstrate the location-dependent network orientations of ICC subgroups and suggest a practical approach for in-depth imaging and quantitative analysis of ICC in the human colon specimen.     

Morphological and morphometrical characteristics of the esophageal intrinsic nervous system in the golden hamster

Very little is known about esophageal innervation in the hamster. In the present study, we used protein gene product 9.5 (PGP 9.5) to determine immunohistochemically the architectural features of the enteric nervous system in the hamster esophagus. The myenteric plexus consisted of a loose and irregular network of ganglia and interganglionic nerve bundles. The density of the neurons in the myenteric plexus was relatively low (479 +/- 75/cm(2), n = 5), with a preferentially higher density in the upper cervical portion than other parts of the esophagus. Regional differences in the number of PGP 9.5-positive neurons and ganglia were observed. PGP 9.5-immunoreactive fibers in the ganglia often branched, giving rise to expanding nerve endings of laminar morphology resembling intraganglionic laminar endings described in rats and cats. Fine varicose fibers originating from the secondary plexus were occasionally observed near the motor endplates, suggested a dual innervation of the striated muscle. The submucosal plexus was free from ganglionated plexus. A regional difference in the submucosal nervous network was observed. The number of motor endplates in the inner muscle layer was higher than that in the outer muscle layer.     

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

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