| In 1893, interstitial cells of Cajal (ICC) were first described in the intestine of rabbit and guinea pig by a great Spanish neuroanatomist, Santiago Ramon y Cajal. On the basis of the staining characteristics of ICC with methylene blue and silver chromate, Cajal believed ICC were a type of terminal of autonomic nerves. After more than a century studies of morphology, ultrastructure, ontogeny and physiology, however, investigators have been certain that ICC were a special interstitial cells forming cellular network in gastrointestinal tract. They play a very important role in the regulation of the gastrointestinal motility by generating and propagating spontaneous electric slow-wave activity and mediating excitatory and inhibitory neurotransmission. Moreover, ICC have been found to be involved in the occurrence of achalasia of cardia, diabetic gastroparesis, chronic intestinal pseudo-obstruction, Hirschsprung's disease, chronic slow-transit constipation and gastrointestinal stromal tumors. Hereby, more and more investigators, including clinical investigators have focused on the field of gastrointestinal kinetics.It had been a long and hard time that scientists could only identify ICC by methylene blue staining, zinc iodide staining and electron microscopy. This limitation in methodology made researchers can neither get further understanding of ICC nor perform their work on development. Fortunately, in the mid-1990s, an amazing specific marker for ICC, c-kit proto-oncogene, was discovered by three research groups led by Huizinga, Maeda and Sanders. C-kit proto-oncogene, an allele of dominant white spotting gene locating in human chromosome 4ql 1-12, encodes type â…¢ receptors tyrosine kinase (KIT) and the ligand for KIT had been identified as stem cell factor(SCF). The continuous expressions of c-kit gene and KIT protein in intramural ICC of gastrointestinal gut were necessary for the development, differentiation, and phenotype maintaining of ICC and the c-kit gene, as well as KIT protein, could be a specific marker of ICC. This discovery not only led to a reliable identification technique for ICC, butalso provided a new tool for further investigation of the origination, development, distribution, physiological function and pathological changes of ICC.Using this special marker, researchers have gained much more information on the structure and function of ICC. However, there are still a lot of mysteries hiding in the fog. For instance, what triggers the development of ICC during embryogenesis, how can the human ICC develop and form a cellular network, how the pacemaker in gastrointestinal tracts were modulated by nerves and what is the relationship between ICC and gastrointestinal motility disorders? In this research, using immunocytochemical stain, NADPH-d cytochemistry and transmission electron microscopy combined with whole mount preparations and cryo-sections, we carefully studied the morphological characters of the above questions, hoping to find new policies and techniques either for researches on the regulation mechanism of gastrointestinal motility or for drugs targeting ICC to treat gastrointestinal motility disorders.Results:I. Development of ICC in human fetal small intestine1. Immunocytochemistry on whole-mount preparations and cryo-section for c-kit and vimentin were adopted in this part. C-kit immunoreactive cells were visualized to be associated with myenteric plexus as early as ten weeks of gestation. Subsequently, we found a few ICC emerged in circular muscle during the mid-stage of gestation and ICC in deep muscular plexus didn't show their existence until the time of birth. These evidences indicated there was a time sequence in the occurrence and development of the different subpopulations of human ICC.2. At early stage of embryogenesis, ICC surrounding intestinal myenteric plexus only formed a single cellular layer with their dominant axis parallel to the circular muscle. With the increase of gestational age, two layers of ICC appeared, which were parallel to circular and longitudinal smooth muscle respectively. However, the fact that this situation could be only found in human indicated there was a species difference in the structure and distribution of ICC. Regulation of automatic rhythm movement in human intestine might be far more complicated than other animals.3. There was also a time order for the formation of ICC network associated with myenteric plexus in human fetal intestine. During the early and middle stages of embryogenesis, there were only a few ICC in spindle-shape with two short processes surroundmyenteric plexus and the cellular network appeared to be disrupted. When it came to the late stage, increasing ICC were nearly mature with more branches and prolonged processes and they connected each other with their processes to form a complete cellular network similar to that of adult, indicating that ICC might possess the function for controlling the gastrointestinal motility before birth. Any delay or abnormal of this development might lead to some gastrointestinal motility disorder in neonate.II. NO innervation on ICC in human fetal small intestine1. A great number of NO neurons and nerves, which constituted the main neuronal component of ganglia, connecting strands, and nerve fiber, were found within the circular muscle layer and myenteric plexus of the small intestine by NADPH-d histochemistry examination. This indicated that NO nerves were fundamentally developed during the middle and late stages of embryogenesis and structurally prepared for the regulation of gastrointestinal motion.2. Double labeling of NADPH-d and KIT immunohistochemistry on whole-mount preparations and cryo-sections revealed a close distribution between NO positive nerves and ICC network surrounding myenteric plexus and some varicosity-like endings around the ICC cell bodies and their processes, which suggested NO nerves in human intestinal myenteric plexus might have some effects on IC-MY.III. Morphological studies on intramural ICC of colon in patients with Hirschsprung's disease and slow-transit constipation1. Observations of transmission electron microscopy and immunohistochemistry on cryo-sections and whole-mount preparations showed statistically significant decrease of all subpopulations of ICC in patients with STC and those ICC locating in the submucosal border (IC-SM) were especially noted for the most significant reduction of 60%. The significant loss of intestinal pacemaker cells may abolish electric slow-wave and lead to the decrease of contractile response and thereby contributed to the prolonged colonic transit time. By this point, we concluded that ICC in the sigmoid colon might be involved in the pathogensis and development of STC.2. A relatively normal distribution of ICC was found in the ganglionic segments of patients with HD. While in aganglionic segments, all subpopulations of ICC decreased and ultrastructure observation showed that organells in ICC decreased and presented regressivepathological changes, indicating the loss of ganglion cells and the reduction of ICC might simultaneously participate in the development of gastrointestinal motility disorders in HD. |
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