Fine structural study of interstitial cells associated with the deep muscular plexus of the rat small intestine,with special reference to the intestinal pacemaker cells

Two types of interstitial cells have been demonstrated in close association in the deep muscular plexus of rat small intestine, by electron microscopy. Cells of the first type are characterized by a fibroblastic ultrastructure, i.e. a well-developed granular endoplasmic reticulum, Golgi apparatus and absence of the basal lamina. They form a few small gap junctions with the circular muscle cells and show close contact with axon terminals containing many synaptic vesicles. They may play a role in conducting electrical signals in the muscle tissue. Cells of the second type are characterized by many large gap junctions that interconnect with each other and with the circular muscle cells. Their cytoplasm is rich in cell organells, including mitochondria, granular endoplasmic reticulum and Golgi apparatus. They show some resemblance to the smooth muscle cells and have an incomplete basal lamina, caveolae and subsurface cisterns. However, they do not contain an organized contractile apparatus, although many intermediate filaments are present in their processes. They also show close contacts with axon terminals containing synaptic vesicles. These gap-junction-rich cells may be regular components of the intestinal tract and may be involved in the pacemaking activity of intestinal movement.     

Failure to demonstrate serotonergic neurons in the myenteric plexus of the rat

Summary Several recent studies suggested that serotonergic neuron-like elements are present in the guinea pig ileum. The present paper reports an extensive study of the digestive tract of the rat with the use of a histofluorescence technique. Administration of the serotonin precursor, tryptophan, associated with a monoamine oxidase inhibitor, did not allow histochemical demonstration of rapidly fading, yellow fluorescent, 6-hydroxydopamine-resistant neurons; conversely such neurons were readily detected in the brain. It is concluded that serotonergic neuron-like elements cannot be detected histochemically in the rat myenteric plexus area after chemical sympathectomy.     

Interstitial cells associated with the deep muscular plexus of the guinea-pig small intestine,with special reference to the interstitial cells of Cajal

Summary Interstitial cells associated with the deep muscular plexus of the guinea-pig small intestine were studied by electron microscopy, and three-dimensional cell models were reconstructed from serial ultrathin sections with a computer graphic system. Three types of cells were recognized. The first type was similar in shape to smooth muscle cells, but did not contain an organized contractile apparatus. Many large gap junctions comprising about 4% of the cell surface were present; they connected cells of the first type to each other, to the second type of cell and to smooth muscle cells of the outer circular layer. The second type of cell had a welldemarcated cell body with long slender processes and was characterized by a large amount of glycogen comprising about 9% of the cell volume. The third type of cell was similar to fibroblasts, and contained well-developed Golgi apparatus and rough endoplasmic retiulum. Some of these fibroblast-like cells (a possible subtype) formed small gap junctions. All three types of cells showed close relationships with nerve varicosities. This cellular network consisting of gap-junction-rich cells, glycogen-rich cells and smooth muscle cells may be involved in the pacemaking activity of intestinal movement.     

The interstitial cells in the colon of the rabbit

Summary The interstitial cells associated with the myenteric plexus of the rabbit colon were studied by scanning and transmission electron microscopy. It was demonstrated that the interstitial cells were stellate or fusiform in shape and located over the ganglia, over nerve bundles and between muscle cells. They were characterized by many slender processes, and resemble fibroblasts. No basal lamina was observed between the interstitial cells and muscle cells. It was concluded that structural features of the interstitial cells are distinctly different from those of neurons, Schwann cells, or of smooth muscle cells, while they show clear similarities to those of fibroblasts. By scanning electron microscopy the shapes and the relations of these cells could be demonstrated in great detail.     

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     

Comparative studies of quinacrine-positive neurones in the myenteric plexus of stomach and intestine of guinea-pig,rabbit and rat

Summary The number of quinacrine-fluorescent nerve cell bodies and the percentage of the ganglion area occupied by this fluorescence within stretch preparations of the myenteric plexus of the stomach and ileum of the guineapig, rabbit and rat were assessed. The number of quinacrine-positive cell bodies per cm² of plexus varied between 1045 in the rabbit ileum to 2633 in the rat stomach, whilst the percentage of the ganglionic area occupied by fluorescence was approximately 10 %. The distribution of quinacrine-fluorescent nerve fibres and cell bodies in the myenteric plexus was compared to the distribution of nerves revealed by catecholamine fluorescence and by staining for acetylcholinesterase in the stomach and ileum of all three species. Quinacrine fluorescence appears to be selective for non-adrenergic, non-cholinergic nerves; the possibility that it binds to high levels of ATP is discussed.     

Three-dimensional demonstration of the interstitial cells of Cajal associated with the submucosal plexus in guinea-pig caecum

The guinea-pig caecum was studied by using immunohistochemistry for Kit receptors and nerves to clarify whether interstitial cells of Cajal (ICC) were localized in association with the submucosal plexus (ICC-SP). A large area of the guinea-pig caecum was nearly devoid of longitudinal muscles, because they were concentrated into three bundles of the taenia caeci (coli) and this allowed clear observation of the myenteric and submucosal plexus as separate networks in whole-mount stretch preparations. The myenteric plexus was observed as a loose polygonal network consisting in elongated ganglia and long connecting nerve strands, whereas the submucosal plexus was identified as smaller ovoid ganglia connected to much thinner nerve strands in different tissue layers. Three-dimensional reconstruction of confocal images revealed multipolar-shaped ICC-SP located around the submucosal ganglion in a basket formation. Bipolar ICC-SP were also observed along the connecting nerve strands of the submucosal plexus. The functional involvement of ICC-SP in mucosal activity is discussed in relation to fluid transportation. This three-dimensional study of ICC-SP thus provides a candidate for the most suitable material available for functional experiments examining the physiological significance of ICC-SP.     

A cytofluorometric study of the myenteric plexus in the guinea pig

Summary The myenteric plexus of the guinea pig ileum was studied in stretch preparations of the longitudinal muscle layer with adherent plexuses, and in freeze-dried transverse sections from the small intestinal wall. Catecholamines and serotonin (5-HT) were visualized according to the Falck-Hillarp technique. Emission spectra from the resulting fluorophores and recordings of their rates of photodecomposition were analysed. Adrenergic nerve terminals showed a slow fluorescence fading rate and a fluorescence spectrum compatible with their known contents of noradrenaline (NA), while the enterochromaffin cells showed a rapid exponential fading and a fluorescence spectrum compatible with their known contents of 5-HT. In order to unmask any low amounts of 5-HT in the neurons of the plexus, analysis of fluorescence parameters at various time intervals after pretreatment with reserpine followed by MAO-inhibition was performed. With the methods used no evidence of the presence of 5-HT in the myenteric plexus of the guinea pig could be found.We thank Iréne Svensson and Uno Johansson for skilful technical assistance. We are also indebted to Ciba, Pfizer and Draco for generous supplies of Reserpine, Nialamide and Pheniprazine. —This work was supported by grants from the Swedish Medical Research Council (Project 14 X-2235) and Göteborgs Läkaresällskap.     

Smooth muscle cells, interstitial cells of Cajal and myenteric plexus interrelationships in the human colon

The plane between longitudinal and circular muscle of human colon, as revealed on examination with light and electron microscopes, has no clear-cut border. Some groups of smooth muscle cells, obliquely oriented and with features similar to both circular and longitudinal ones--the connecting muscle bundles--run from one muscle layer to another. Other groups of smooth muscle cells, possessing their own specific ultrastructural features--the myenteric muscle sheaths--, make up envelopes of variable thickness around some myenteric ganglia and nerve strands, partially or completely embedding them in one or other muscle layer. Non-neuronal, non-muscular cells (interstitial cells of Cajal, covering cells, fibroblast-like and macrophage-like cells) complicate the texture of the myenteric muscle sheaths, creating an intricate, interconnected cellular network inside them, widespread among nerve bundles and smooth muscle cells; however, only interstitial cells have cell-to-cell junctions also with the smooth muscle cells and nerve endings. These data document the existence in this colonic area of two different types of muscle cell arrangements, one of which, the myenteric muscle sheath, only contains putative pacemaker cells.     

Possible involvement of muscularis resident macrophages in impairment of interstitial cells of Cajal and myenteric nerve systems in rat models of TNBS-induced colitis

Resident macrophages are distributed in the network of interstitial cells of Cajal (ICC) and the myenteric nerve within the myenteric plexus. We evaluated changes in chemoattractant protein mRNA expression in macrophages and neutrophils, the ICC, nerve and macrophages in the myenteric plexus of model rats with TNBS-induced colitis. Chemoattractant proteins, MCP-1, GRO, MIP-2 and CINC-2α were upregulated in the colonic muscle layer after inflammation. Leukocyte infiltration and MPO activity were increased in the muscle layer. Electron microscopy indicated an irregular contour of the myenteric ganglia into which numerous macrophages had penetrated. Macrophages were also distributed near the ICC in the inflamed myenteric plexus. Immunohistochemistry showed that the ICC network and myenteric nerve system had disappeared from the inflamed region, whereas the number of resident macrophages was increased. TTX-insensitive, possibly ICC-mediated, rhythmic contractions of circular smooth muscle strips and enteric neuron-mediated TTX-sensitive peristalsis in the whole proximal colon tissue were significantly inhibited in the inflamed colon, indicating that the ICC-myenteric nerve system was dysfunctional in the inflamed muscle layer. Their accumulation around the myenteric nerve plexus and the ICC network suggests that macrophages play an important role in inducing intestinal dysmotility in gut inflammation.     

Ultrastructural observations of the tunica muscularis in the small intestine of Xenopus laevis, with special reference to the interstitial cells of Cajal

The distribution and ultrastructure of the interstitial cells of Cajal (ICC) has been examined in the small intestine of the frog Xenopus laevis, as the physiological significance of these cells remains obscure in amphibians and other lower vertebrates. The present study has revealed the existence of a special type of interstitial cell in the tunica muscularis of the small intestine of Xenopus; this cell is characterized by the presence of numerous caveolae, many small mitochondria, and the formation of intercellular connections with the same type of cell. Since these ultrastructural features are shared with mammalian ICC, the cells in the small intestine of Xenopus probably correspond to ICC. These cells also form close contacts with neighboring smooth muscle cells and with nerve varicosities containing accumulations of synaptic vesicles. These cellular networks are likely to be involved in the transmission of nerve impulses to muscle cells, as has been suggested for mammalian tissues. However, true gap junctions have not been detected; they occur neither between the same type of cells nor between the putative ICC and smooth muscle cells. The widespread distribution of ICC or equivalent cells in different groups of vertebrates, together with the conservation of their ultrastructural features, suggests that they differentiated early in vertebrate evolution to play key regulatory roles in gastrointestinal movement.     

Direct and indirect innervation of smooth muscle cells of rat stomach,with special reference to the interstitial cells of Cajal

Interstitial cells of Cajal in the circular (ICC-CM) and longitudinal (ICC-LM) muscle layer of the rat gastric antrum and their innervation were studied ultrastructurally. Both ICC-CM and ICC-LM are characterized by many mitochondria, rough and smooth endoplasmic reticulum, caveolae, and formation of gap junctions with each other and with muscle cells, though ICC-LM tend to show more variable cytoplasmic features depending on section profiles. Close contacts between nerve terminals and both ICC-CM and ICC-LM are observed. These possible synaptic structures are characterized by: (1) accumulation of synaptic vesicles in nerve varicosities, (2) a narrow gap (about 20 nm) between pre- and postjunctional membranes, (3) lack of a basal lamina between pre- and postjunctional membranes, and (4) the presence of an electron-dense lining on the inner aspect of prejunctional membranes. Almost the same characteristics are observed between the nerve terminals and the muscle cells of both circular and longitudinal muscle layers of the same specimens. Therefore, we conclude that the smooth muscle cells of both circular and longitudinal layers of the rat antrum are directly and indirectly innervated via ICC. Their functional significance is discussed.     

Uptake of horseradish peroxidase from CSF into the choroid plexus of the rat,with special reference to transepithelial transport

Summary Protein uptake from cerebral ventricles into the epithelium of the choroid plexus, and transport across the epithelium were studied ultrastructurally in rats. Horseradish peroxidase (HRP, MW 40,000) was used as protein tracer. Steady-state ventriculo-cisternal perfusion with subatmospheric pressure (-10cm of water) in the ventricular system was applied. HRP dissolved in artificial CSF was perfused from the lateral ventricles to cisterna magna for various times, and ventriculo-cisternal perfusion, vascular perfusion or immersion fixation with a formaldehyde-glutaraldehyde solution was performed.Coated micropinocytic vesicles containing HRP were seen both connected with the apical, lateral and basal epithelial surface and within the cells. Heavily HRP-labeled vesicles were often fused with the lining membrane of slightly labeled or unlabeled intercellular spaces. Since the apical tight junctions of the epithelium never appeared open or never contained HRP in the spaces between the fusion points, and since the intercellular spaces between adjacent epithelial cells below the junctions only infrequently contained tracer after 5 min, by increasing amounts after 15–60 min of HRP perfusion, a vesicular transport of HRP from the apical epithelial surface to the intercellular spaces, bypassing the tight junctions, is suggested.In addition to the transepithelial transport, micropinocytic vesicles also transported HRP to the lysosomal apparatus of the epithelial cells. With increasing length of exposure to HRP, a sequence of HRP-labeled structures could be evaluated, from slightly labeled apical vacuoles and multivesicular bodies to very heavily labeled dense bodies.     

Structure of the tertiary component of the myenteric plexus in the guinea-pig small intestine

The tertiary component of the myenteric plexus consists of interlacing fine nerve fibre bundles that run between its principal ganglia and connecting nerve strands. It was revealed by zinc iodide-osmium impregnation and substance P immunohistochemistry at the light-microscope level. The plexus was situated against the inner face of the longitudinal muscle and was present along the length of the small intestine at a density that did not vary markedly from proximal to distal. Nerve bundles did not appear to be present in the longitudinal muscle as judged by light microscopy, although numberous fibre bundles were encountered within the circular muscle layer. At the ultrastructural level, nerve fibre bundles of the tertiary plexus were found in grooves formed by the innermost layer of longitudinal smooth muscle cells. In the distal parts of the small intestine, some of these nerve fibre bundles occasionally penetrated the longitudinal muscle coat. Vesiculated profiles in nerve fibre bundles of the tertiary plexus contained variable proportions of small clear and large granular vesicles; they often approached to within 50–200 nm of the longitudinal smooth muscle cells. Fibroblast-like cells lay between strands of the tertiary plexus and the circular muscle but were never intercalated between nerve fibre varicosities and the longitudinal muscle. These anatomical relationships are consistent with the tertiary plexus being the major site of neurotransmission to the longitudinal muscle of the guinea-pig small intestine.     

Ultrastructural studies of the myenteric plexus and smooth muscle in organotypic cultures of the guinea-pig small intestine

External muscle and myenteric plexus from the small intestine of adult guinea-pigs were maintained in vitro for 3 or 6 days. Myenteric neurons and smooth muscle cells from such organotypic cultures were examined at the electron-microscopic level. An intact basal lamina was found around the myenteric ganglia and internodal strands. Neuronal membranes, nuclei and subcellular organelles appeared to be well preserved in cultured tissues and ribosomes were abundant. Dogiel type-II neurons were distinguishable by their elongated electron-dense mitochondria, numerous lysosomes and high densities of ribosomes. Vesiculated nerve profiles contained combinations of differently shaped vesicles. Synaptic membrane specializations were found between vesiculated nerve profiles and nerve processes and cell bodies. The majority of nerve fibres were well preserved in the myenteric ganglia, in internodal strands and in bundles running between circular muscle cells. No detectable changes were found in the ultrastructure of the somata and processes of glial cells. Longitudinal and circular muscle cells from cultured tissue had clearly defined membranes with some close associations with neighbouring muscle cells. Caveolae occurred in rows that ran parallel to the long axis of the muscle cells. These results indicate that the ultrastructural features of enteric neurons and smooth muscle of the guinea-pig small intestine are well preserved in organotypic culture.     

Multiple mechanisms contribute to myenteric plexus ablation induced by benzalkonium chloride in the guinea-pig ileum

Ablation of rat myenteric plexus with benzalkonium chloride has provided a model of intestinal aganglionosis, but the degenerative responses are not well understood. We examined the effects of this detergent on neurons and glia, including expression of c-Myc, c-Jun, JunB, and c-Fos, and on immunocytes in the guinea-pig ileum. Benzalkonium chloride (0.1%) or saline was applied to the serosal surface of distal ileum. Tissues were analyzed 2, 3, or 7 days later and compared with cyclosporine-treated and untreated animals. More than 90% of myenteric neurons were destroyed in ileal segments 3–7 days after benzalkonium-chloride treatment. Glia withdrew processes from around neurons after 2 days and were mostly gone after 3 days. Neuronal c-Myc began to disappear while c-Fos, c-Jun, and JunB were evident in some neuronal nuclei after 2 or 3 days. After 3 days, widespread apoptosis was evident in the myenteric plexus. Populations of T cells, B cells, and macrophage-like cells in untreated and saline-treated myenteric plexuses were substantially increased 3 and 7 days after benzalkonium-chloride treatment. Cyclosporine delayed significant neuronal loss. We conclude that a variety of degenerative mechanisms may be active in this model, including an immune response which may actively contribute to tissue destruction. Received: 13 September 1996 / Accepted: 20 January 1997     

Ultrastructure of rat ovarian interstitial gland cells during pregnancy

Summary The fine structure of the interstitial gland of the rat ovary was studied at estrus and on Days 4, 6, 10, 14 and 18 of pregnancy. At estrus, ovarian interstitial cells have small nuclei with dense irregular clumps of heterochromatin. Mitochondria are small and rod-shaped and have predominantely lamellar cristae. Numerous osmiophilic lipid droplets are present. At Days 4 and 6, nuclear heterochromatin is reduced, and nucleoli are larger and complex. Mitochondria are enlarged and often bizarre-shaped and have tubular cristae. Golgi and smooth endoplasmic reticulum are more conspicuous. At Day 10, prominent ultrastructural features include nuclei with conspicuous heterochromatin, smaller mitochondria with both lamellar and tubular cristae, numerous ribosomes and lipid droplets with decreased osmiophilia. At Days 14 and 18, nuclei have increased heterochromatin, mitochondria are small and have lamellated cristae and an increase in the size and number of lipid droplets occurs. These observations suggest that steroidogenic activity of interstitial cells is highest during the first half of pregnancy and regresses during the last half. It is suggested that the interstitial gland is an important ovarian source of pregnancy hormone(s) during the first half of gestation and that LH may modulate steroidogenic activity in this ovarian component.     

Unique distribution of interstitial cells of Cajal in the feline pylorus

The feline gastrointestinal (GI) tract is an important model for GI physiology but no immunohistochemical assessment of interstitial cells of Cajal (ICC) has been performed because of the lack of suitable antibodies. The aim of the present study was to investigate the various types of ICC and associated nerve structures in the pyloric sphincter region, by using immunohistochemistry and electron microscopy to complement functional studies. In the sphincter, ICC associated with Auerbach’s plexus (ICC-AP) were markedly decreased within a region of 6–8 mm in length, thereby forming an interruption in this network of ICC-AP, which is otherwise continuous from corpus to distal ileum. In contrast, intramuscular ICC (ICC-IM) were abundant within the pylorus, especially at the inner edge of the circular muscle adjacent to the submucosa. Similar distribution patterns of nerves positive for vesicular acetylcholine transporter (VAChT), nitric oxide synthase (NOS) and substance P (SP) were encountered. Quantification showed a significantly higher number of ICC-IM and the various types of nerves in the pylorus compared with the circular muscle layers in the adjacent antrum and duodenum. Electron-microscopic studies demonstrated that ICC-IM were closely associated with enteric nerves through synapse-like junctions and with smooth muscle cells through gap junctions. Thus, for the first time, immunohistochemical studies have been successful in documenting the unique distribution of ICC in the feline pylorus. A lack of ICC-AP guarantees the distinct properties of antral and duodenal pacemaker activities. ICC-IM are associated with enteric nerves, which are concentrated in the inner portion of the circular muscle layer, being part of a unique innervation pattern of the sphincter. This study was supported by operating grants from the Canadian Institutes of Health Research (to J.D.H. and N.E.D.) and from the Canadian Association of Gastroenterology (to L.W.C.L.).     

Trypanosoma cruzi: preliminary investigation of NADH-positive and somatostatin-immunoreactive neurons in the myenteric plexus of the mouse colon during the infection

In this paper, the distribution of NADH-positive and somatostatin (SOM) immunoreactive neurons in the myenteric plexus of the colon of mice infected with Trypanosoma cruzi was studied. Ten young, male, BALB/c mice were inoculated with the Y strain of T. cruzi, 60 days previously (chronic phase of the infection). Another 10 mice were uninfected controls. Distal and proximal colonic neurons from five chronically infected mice and their controls were stained using the NADH-diaphorase method. Quantitative results showed a significant decrease of 39% in the number of neurons in the proximal colon of infected mice and 58% in the distal colon (p<0.05). SOM was localized in five animals from each group by light microscopy, using an indirect immunofluorescence technique. It was observed that there were far fewer nerve cells and fibres and less intensely stained neuron bodies and varicose SOM-positive nerve fibres in both, control and chronic infected mice. These findings could be related to the disturbances in intestinal motility observed in patients in the chronic phase of Chagas' disease.