Ichiro Takayama

Loss of interstitial cells of Cajal and development of electrical dysfunction in murine small bowel obstruction

1 Partial obstruction of the murine ileum led to changes in the gross morphology and ultrastructure of the tunica muscularis. Populations of interstitial cells of Cajal (ICC) decreased oral, but not aboral, to the site of obstruction. Since ICC generate and propagate electrical slow waves in gastrointestinal muscles, we investigated whether the loss of ICC leads to loss of function in partial bowel obstruction. 2 Changes in ICC networks and electrical activity were monitored in the obstructed murine intestine using immunohistochemistry, electron microscopy and intracellular electrophysiological techniques. 3 Two weeks following the onset of a partial obstruction, the bowel increased in diameter and hypertrophy of the tunica muscularis was observed oral to the obstruction site. ICC networks were disrupted oral to the obstruction, and this disruption was accompanied by the loss of electrical slow waves and responses to enteric nerve stimulation. These defects were not observed aboral to the obstruction. 4 Ultrastructural analysis revealed no evidence of cell death in regions where the lesion in ICC networks was developing. Cells with a morphology intermediate between smooth muscle cells and fibroblasts were found in locations that are typically populated by ICC. These cells may have been the redifferentiated remnants of ICC networks. 5 Removal of the obstruction led to the redevelopment of ICC networks and recovery of slow wave activity within 30 days. Neural responses were partially restored in 30 days. 6 These data describe the plasticity of ICC networks in response to partial obstruction. After obstruction the ICC phenotype was lost, but these cells regenerated when the obstruction was removed. This model may be an important tool for evaluating the cellular/molecular factors responsible for the regulation and maintenance of the ICC phenotype.

Immunolocalization of dystrobrevin in the astrocytic endfeet and endothelial cells in the rat cerebellum

Dystrobrevin is a newly discovered dystrophin-associated protein that is classified as alpha- and beta-dystrobrevin. Previous studies reported that dystrophin, utrophin, syntrophin and beta-dystroglycan were expressed in the cerebellum. In the present study, we examined cellular and subcellular localization of dystrobrevin in the adult rat cerebellum immunohistochemically. Confocal microscopy showed that dystrobrevin was expressed around blood vessels and under the pia mater as dystrophin, utrophin and beta-dystroglycan were. Immunoelectron microscopy demonstrated that dystrobrevin was localized not only in the astrocytic endfeet around blood vessels and under the pia mater, but also in endothelial cells. Considering the fact that dystrobrevin possesses multiple phosphotyrosine kinase residues, these data suggest that dystrobrevin plays a role in blood-brain barrier functions as a component of the dystrophin complex.

Differential gene expression in the murine gastric fundus lacking interstitial cells of Cajal

BackgroundThe muscle layers of murine gastric fundus have no interstitial cells of Cajal at the level of the myenteric plexus and only possess intramuscular interstitial cells and this tissue does not generate electric slow waves. The absence of intramuscular interstitial cells in W/WVmutants provides a unique opportunity to study the molecular changes that are associated with the loss of these intercalating cells.MethodThe gene expression profile of the gastric fundus of wild type and W/WVmice was assayed by murine microarray analysis displaying a total of 8734 elements. Queried genes from the microarray analysis were confirmed by semi-quantitative reverse transcription-polymerase chain reaction.ResultsTwenty-one genes were differentially expressed in wild type and W/WVmice. Eleven transcripts had 2.0–2.5 fold higher mRNA expression in W/WVgastric fundus when compared to wild type tissues. Ten transcripts had 2.1–3.9 fold lower expression in W/WVmutants in comparison with wild type animals. None of these genes have ever been implicated in any bowel motility function.ConclusionsThese data provides evidence that several important genes have significantly changed in the murine fundus of W/WVmutants that lack intramuscular interstitial cells of Cajal and have reduced enteric motor neurotransmission.

Differential gene expression in the small intestines of wildtype and W/WV mice

Much of the evidence demonstrating the role of interstitial cells of Cajal (ICC) in pacemaking and neurotransmission in the gastrointestinal tract comes from studies of W/WV mice. These animals have few pacemaker ICC in the small bowel due to reduced functional Kit protein. We examined gene expression in the small intestines of wildtype and W/WV mice. RNA expression in the jejunums of wildtype and W/WV mutants was studied using a differential gene expression method. Seven known genes were differentially expressed in wildtype and W/WV mice. COX7B (cytochrome c oxidase, subunit VIIb) and SORCIN (encoding multidrug‐resistance complex, class 4) were suppressed in both fed and fasted W/WV mice. Expression of another five genes was increased in W/WV mice: ADA (adenosine deaminase), MDH1 (malate dehydrogenase), RPL‐8 (ribosomal protein L8), SPTB2 (spectrin, nonerythroid, beta subunit), and p6–5 (encoding phosphorylcholine [PC] T‐cell suppressor factor [TsF]). Differential expression was the same in fasted and fed animals, suggesting that the differences were independent of the dietetic state. We conclude that several genes are differentially expressed in the small intestines of W/WV mice where the major lesion is loss of pacemaker ICC. Differential gene display may help develop a molecular profile of motility disorders in which ICC are lost.

Immunoelectron-microscopic study of Kit-expressing cells in the jejunum of wildtype and Ws/Ws rats.

Abstract. Interstitial cells of Cajal (ICC) are responsible for generating electrical slow waves in the gastrointestinal (GI) tract. Slow waves regulate the frequency of contractions of the tunica muscularis, and therefore ICC are critical for normal motility in the small intestine. ICC express Kit, the gene product of c-kit, a protooncogene that encodes a receptor tyrosine kinase. Physiological evidence demonstrating that ICC are pacemakers has come from experiments on W-mutant mice which have few Kit-positive cells at the level of the myenteric plexus (IC-MY) and also lack electrical slow waves. In the past identification of ICC required the use of electron microscopy, however the discovery that ICC express Kit has facilitated studies of the distribution of ICC in several species. Immunoelectron microscopy to relate ultrastructure to Kit expression has only been performed in a limited number of studies of mice. We examined the ultrastructure of Kit-expressing cells in the rat using immunoelectron microscopy and an anti-Kit antibody. We compared the presence and appearance of Kit-expressing ICC in wildtype and Ws/Ws rats, which carry a mutation in the white spotting locus and have a phenotype similar to W/WV mutant mice. Kit-expressing cells could be detected in the myenteric plexus (MY) and deep muscular plexus (DMP) regions of the small intestine of wildtype animals. In Ws/Ws rats, Kit-expressing cells were not observed in the region of MY, but were observed in the DMP. The density of Kit-positive cells in the DMP of Ws/Ws rats was similar to those in wildtype rats. Electron microscopy showed that Kit-expressing cells at the level of the MY of the rat had similar ultrastructural features as IC-MY in wildtype mice. IC-DMP in the rat of both wildtype and Ws/Ws mutants were similar in structure to IC-DMP of the mouse. We conclude that wildtype rats have IC-MY and IC-DMP in the tunica muscularis of the jejunum. ICC express Kit-like immunoreactivity (Kit-LI) in the rat as in the mouse. IC-MY are absent in the small intestine of Ws/Ws rats, and this corresponds to the lack of Kit-labeling in this region. Ws/Ws rats, however, possess IC-DMP with normal ultrastructural features and Kit-LI. The absence of IC-MY of Ws/Ws rats is likely to account for the abnormal contractile activity of the GI tract observed in these mutants. The present study suggests that Ws/Ws rats could provide an interesting model to investigate the physiological significance of pacemaker activity because they manifest a defect in IC-MY.

Ultrastructure of rat duodenal myenteric plexus revealed by quick-freezing and deep-etching method.

Abstract: A quick-freezing and deep-etching (QF-DE) method was employed with whole-mount strips of rat duodenal muscle walls to exhibit the cytoskeletons of the myenteric plexus. Nerve fibers in the myenteric plexus, which contained fewer neurofilaments than other types of neurons examined, had many varicosed contours, and were bundled by enteroglial cells. Cytoskeleton arrays were rarely observed in the varicosed regions, where synaptic vesicles were often seen, although other nerve regions contained many neurofilaments running almost in parallel with the nerve fiber bundle. Enteroglial cells had short cytoskeletons predominantly across the cytoplasm, becoming thinner the around varicosed regions of the nerve bundles. Such enteroglial extruded areas were often in close association with neighboring nerve fibers, indicating intercommunications between the nerve fibers. In distal parts of enteric nerve processes, there were numerous synaptic vesicles, but few neurofilaments. Smooth muscle cells were closely associated with the enteric nerve processes. Fine network structures, responsible for the extracellular matrix, were present between the smooth muscle cells and the enteric nerve processes. These specific structures of the myenteric plexus could be important for signalling or for the transportation of neurotransmitters involved in gut motility.

Novel human and mouse genes encoding a shank‐interacting protein and its upregulation in gastric fundus of W/WV mouse

Background and Aims: A division of labor exists between different classes of interstitial cells of Cajal (ICC) in the gastrointestinal tract. In the stomach and small intestine, ICC at the level of the myenteric plexus (IC‐MY) act as slow wave pacemaker cells, whereas intramuscular ICC (IC‐IM) in the stomach act as intermediaries in enteric motor neurotransmission. The muscle layers of the gastric fundus do not have IC‐MY, therefore electric slow waves are not generated. Intramuscular ICC are absent in the gastric fundus of W/WV mutant mice, and excitatory and inhibitory motor nerve responses are reduced in these tissues. The absence of IC‐IM in W/WV mutants in the fundus provides a unique opportunity to study the molecular changes that are associated with the loss of these cells.

Dynamic ultrastructure of mouse pulmonary alveoli revealed by an in vivo cryotechnique in combination with freeze-substitution

A morphological approach to cell dynamics is usually difficult, since routine preparative techniques for electron microscopy always induce artifacts due to cessation of the blood supply into organs. An in vivo cryotechnique followed by the freeze‐substitution method probably reduces such problems. It was applied for examining the pulmonary alveoli of BALB/c mice in vivo. The following ultrastructural features were revealed. (1) A surfactant layer provided a continuous covering to the alveolar epithelium. (2) Pleural epithelial cells, alveolar cells and endothelial cells contained many small vesicles and pits. In the alveolar epithelium, they were often localised near microtubules. (3) Typical lamellar structures in large alveolar epithelial cells were rarely detected. (4) Circulating erythrocytes with various shapes were observed in branching blood capillaries. (5) A close association between erythrocytes and the endothelium was seen at the peripheral alveolar septum. Such ultrastructural arrangements may be appropriate for the physiological functions of the pulmonary alveoli, such as exchanges of gases or materials in vivo.

REVIEW: Changes of In Vivo Gastrointestinal Motor Pattern in Pacemaker-Deficient (WsRC-Ws/Ws) Rats

In vitro studies on pacemaker-deficient W-mutants have revealed a disappearance of rhythmic contraction in their gastrointestinal tracts. Their contractile force has not been diminished, however. In contrast, W-mutants often present dysmoility-like symptoms with distension of the gastrointestinal tract in vivo. Gastrointestinal motility of W-mutant rats was examined in vivo by an extraluminal strain-gauge force transducer method. We examined a normal gastrointestinal motor pattern in the rats with two distinct motor phases, digestive and interdigestive. Moreover, we detected a failure to form an interdigestive contractile complex in pacemaker-deficient rats. The interdigestive motor activity of the gastrointestinal tract is important for cleaning gastrointestinal tract in preparation for the next meal. The impairment of the interdigestive contractile complex may be related to the dysmoility-like symptoms of W-mutant rats in vivo.

Differential gene expression profile in the small intestines of mice lacking pacemaker interstitial cells of Cajal

BackgroundWe previously identified eight known and novel genes differentially expressed in the small intestines of wild type and W/WVmice, which have greatly reduced populations of the interstitial cells of Cajal, that are responsible for the generation of electrical slow waves, by using a differential gene display method.MethodsBy using the same method we isolated additional candidate genes that were specifically down- or up-regulated in W/WVmice. Novel transcripts were designated as DDWMEST.ResultsWe isolated seven candidates that were specifically down- or up-regulated in W/WVmice. Two novel transcripts, DDWMEST 1 and -91 were increased in both fed and fasted W/WVmice. Expression of another five genes was suppressed in W/WVmice: ARG2 (Arginase II), ONZIN (encoding leukemia inhibitory factor regulated protein), and three novel transcripts: DDWMEST62, -84, and -100. Together with the previous report, we identified fifteen differentially expressed genes in total in the small intestines of W/WVmice. Eight of these genes were reduced in the jejunums of W/WVmice compared to age matched wild type mice, whereas the other seven genes showed an increase in expression. Differential expression was the same in fasted and fed animals, suggesting that the differences were independent of the dietetic state of the animal.ConclusionsSeveral known and novel genes are differentially expressed in the small intestines of W/WVmice. Differential gene comparison might contribute to our understanding of motility disorders associated with the loss of the interstitial cells of Cajal.