Studies On Characteristics Of The Interstitial Cells Of Cajal And The Enteric Nervous System In The Chicken Intestine

At present, it is generally accepted that the normal intestinal peristalsis is dependent on the interaction among the enteric nervous system (ENS), the interstitial cells of Cajal (ICC) and smooth muscle cells. ICC is a special class of interstitial cells located between the autonomous nerve endings and gastrointestinal smooth muscle cells. Works mainly in three aspects:The ICC generate pacemaker activity throughout the gastrointestinal tract by initiating the slow wave activity that is integral to the coordination of gastrointestinal motility; In addition, ICC are thought to be involved in mediating neurotransmission and facilitating active propagation of electrical events.In the past, the studies on ENS and ICC are mainly concentrated in the mammals’ gastrointestinal tract. The structure between birds and mammals is different, and the ENS of avian contains neurons that are in the wall of the gastrointestinal tract, as well as the intestinal nerve of Remak (INR) located in the mesenteric edge outside of the intestinal wall. Would this variation lead to the difference of the structure and mechanism of interactions between the ENS and the ICC? Chicken was selected as experimental animals on behalf of birds for detail researches of gastrointestinal tract, such as the identification and distribution of ICC, the relations between ICC and nerves and smooth muscle. In addition, the comparative study on the postnatal development and distribution of NOS and AChE positive neurons were revealed, because the two types of neurons were closely related with ICC in intestine. Those researches explain the structure and relation of cells and molecule in the ENS and ICC from chicken intestine, and analyze the functional significance of the ENS and ICC. This study provide basic datum for the works of the function and the protection of diseases in chicken gastrointestinal tract. Experiment I Identification and distribution of interstitial cells of cajal in the intestine of chicken The present study has observed the ultrastructural feature of ICC in the chicken intestine by transmission electron microscopy (TEM). The results showed ICC could be found in different regions and the numbers were different. They were classified into several subtypes:ICC at the layer of myenteric plexus、ICC at the intramuscular level、 ICC in the deep muscular plexus、ICC on the submucosal plexus and ICC in the lamina propria mucosae. This cells share common ultrastructural features, such as the presence of numerous mitochondria, abundant intermediate filaments, their nuclei were fusiform, oval or indented with a dense band of peripheral heterochromatin, they also form close contacts with neighbouring SMC (SMC) and with enteric nerve. They are spindle-shaped or stellate, having a different number of ramified cell processes. In addition, some ICC also shows some typical features of SMC including a basal lamina, caveolae, and dense bodies. Ture gap junctions have been detected between with other ICC or with neighbouring SMC. We also identified a possible new subtype of ICC located in the lamina propria mucosae, which was different with mammals. Fibroblast-like cells (FLC) were abundant showing well-developed secretory organelles, including coated vesicles, a patchy basal lamina, but lacked prominent intermediate filaments and caveolae. The widespread distribution of ICC or equivalent cells in chicken intestine, together with the conservation of their ultrastructural features, suggests that they play same key regulatory roles in gastrointestinal movement with mammals. The observation of ICC in the lamina propria mucosae indicates that there may be more subtypes of these cells in the intestine than previously considered.Experiment II The identification and expression of c-kit positive cells in the intestine of chicken The distribution of the interstitial cells of Cajal (ICC) remains obscure because of lacking suitable marker. In the present study on the basis of the third chapter about the ultrastructure identification of ICC in chicken intestine, the identification and expression of c-kit positive cells in the chicken intestine were demonstrated by means of in situ hybridization (ISH) histochemistry, and the expression of c-kit gene in the intestinal wall lacking mucosa and mucosa of normal chickens were also revealed by real-time fluorescence quantitative polymerase chain reaction (RT-PCR). Two types of cells stained positive for c-kit mRNA. The first group consisted of spindle-shaped or bipolar cells identified as ICC. ICC were found at a variety of locations-at the level of the myenteric plexus between the circular and longitudinal muscle, and intermingled with smooth muscle cells within muscle bundles in the circular and longitudinal muscle layers. ICC was also identified along the submucosal layer; they were classified into several subtypes. The second group was comprised of round-shaped cells, which resembled mast cells. Mast cells were mainly found in the lamina propria region as well as in submucosal layer. RT-PCR revealed the expression of c-kit mRNA throughout the intestinal wall lacking mucosa and mucosa of the total intestine. In intestinal wall lacking mucosa, c-kit mRNA was detected at high levels in all samples of the ileum and colon, moderate levels in the duodenum and jejunem, low levels in the caeca; in mucosa, c-kit mRNA was detected at high levels in all samples of the jejunum, following by ileum and duodenum. While the levels of mRNA in the caeca and colon was low. Conclusively, this study reveals for the first time the distribution of ICC, quantifies the expression of c-kit mRNA in the intestine of chicken and also compares the c-kit positive cell types on morphologically. The results indicate the difference may be related to different bowel functions.Experiment Ⅲ The study on c-kit and SCF mRNA expression in chicken intestine of different ages Tissue samples (the intestinal wall lacking mucosa of duodenum, jejunum, ileum, caeca, and colon) were made in1-days-old,10-days-old,20-days-old and adult (40-days-old) chickens (n=5) and identify the expression of c-kit and its ligand SCF mRNA in this tissues by real-time fluorescence quantitative polymerase chain reaction (RT-PCR) with SYBR green and were normalized against the housekeeping gene β-actin. The results showed that the expression patterns were different between c-kit and SCF genes. The expression of c-kit mRNA was decreased in different intestinal segments with ages. In1-days-old group, c-kit mRNA was detected at highest levels, the expression of c-kit mRNA detected in20-days-old and adult group were extremely significantly lower than that of1-days-old group. The expression of SCF mRNA was increased from1-days-old to10-days-old, and then the tendency was descend. The expression of SCF mRNA detected in20-days-old and adult group were significantly lower than that of10-days-old group. In different age groups, the expression of c-kit and SCF mRNA showed a dynamic change. In1-days-old group, c-kit mRNA was detected at highest level in colon and lowest level in caeca, SCF mRNA was detected at highest level in duodenum and lowest level in caeca. In10-days-old group, c-kit mRNA was detected at highest level in colon and lowest level in jejunum, SCF mRNA was detected at highest level in colon and lowest level in caeca. But in20-days-old and adult group, the change was similar, SCF and c-kit mRNA was detected at highest level in ileum and lowest level in caeca. Conclusively, this study reveals it is a close relationship between SCF and c-kit and indicates the functional maturation in development of postnatal intestine is a gradually process.Experiment Ⅳ Double-labeled immunofluorescence study on vimentin and NSE protein in chicken intestine Different intestinal segments were used in this study in order to investigate the morphological relationship between the interstitial cells of Cajal and nerve using immunofluorescent study on vimentin and Neuron-specific Enolase (NSE) by double-labeled immunofluorescence combined laser-scanning confocal microscopy, and distribution of the two positive cells were analyzed. The results showed vimentin positive cells and their processes were mainly found within lamina muscularis, submucosal layer and lamina propria, specially in he myenteric plexus between the circular and longitudinal muscle. However, vimentin positive cells were distributed in the septum of lamina muscularis of the large intestine and within the muscle bundles of small intestine. NSE positive cells mainly located in the myenteric plexus between the circular and longitudinal muscle and submucosal layer, and scattered within muscle bundles in the circular and longitudinal muscle layers. NSE positive cells were lower in mucosa than that of lamina muscularis, and these cells were more in small intestinal mucosa than that of large intestine; these cells were also found near mucosal epithelium. We found the vimentin positive cells closely surround the NSE positive cells in lamina muscularis and submucosal layer by double-labelling, and similar relations seen in mucosa. Conclusively, the vimentin and NSE positive cells form close contacts with each other, suggesting they play an important biological role in the intestinal motility.Experiment V The structural study on NOS positive neurons in myenteric plexus of the chicken intestine during postnatal development Information regarding the development of the enteric nervous system (ENS) is important for understanding the intestinal function. Because fertilized chicken eggs provide easy access to embryos chicken model have been widely used to study developing fetal myenteric plexus, however no study has been focused on the postnatal period. The aim of this study was to perform a qualitative and quantitative analysis of NOS positive cells (the nitrergic neurons) in the myenteric plexus of developing chickens in the postnatal period. Whole-mount preparations of the myenteric plexus were made in7-days-old,15-days-old and adult chickens (n=15). The myenteric plexus was studied after nicotinamide adenine dinucleotide phosphate diaphorase (NADPH-d) histochemistry using light microscopy, digital photography and IPP6.0software. The number of positively stained neurons and ganglia was counted in the duodenum, jejunum, ileum, cecum and colon in the different age groups. The results showed the nerve fiber bundles connecting the ganglia form a clear meshwork. The positively stained neurons showed various morphology, staining intensity and formed bead-shaped and U-shaped arrangements in the myenteric plexus. The number of positively stained neurons per area and the number of ganglia per area decreased with age; however the number of positive neurons per ganglia increased. The number of NADPH-d positive neurons was highest in the colon, following by the ileum, the jejunum, the duodenum and the cecum in all age groups. Conclusively, Developmental changes in the myenteric plexus of chickens continue in the postnatal period, this indicates that the formation of function needs a gradual process of maturation and improvement. In addition, no significant difference is seen among different intestinal segments during postnatal development, suggesting that the function of different intestinal segments had been determined after birth.Experiment VI The study on morphology and distribution of AChE positive neurons in submucosal plexus of the chicken ileum Acetylcholine is recognized as a potent mediator of excitatory nerves responsible for the constriction of the smooth muscle of the gastrointestinal tract, enhance the secretion of the gastrointestinal tract, and stimulate the release of gut hormones. The aim of this study was to determine the normal cholinergic neuronal density and morphology in the submucosal plexus of the chicken ileum from0--days-old to40-days-old. In this experiment, whole-mount preparations of the submucosal plexus of chicken ileum were made in0-days-old,5-days-old,10-days-old,20-days-old and40-days-old chickens. The submucosal plexus was studied after acetylcholine esterase (AChE) histochemistry. The results showed that the positively stained neurons showed various morphology, staining intensity and formed a clear network by ganglia and nerve fibers. The number of ganglia per area decreased with age, however the size of positive neurons increased, and the size in40-days-old group was3times than that of0-days-old group. The change of number of positive neurons per ganglia was dynamic, but the tendency was stably increased from10-days-old to adult. Conclusively, developmental changes in the submucosal plexus of chickens was marked in the postnatal period by qualitative and quantitative analysis, this study will provide an important histopathological reference on the diagnosis of causes of enteric nervous system disorders. Experiment Ⅶ Histochemical localization of the cholinergic and nitrergic neurons in the chicken ileum Histochemical localization and analysis of the cholinergic and nitrergic neurons in the chicken ileum were investigated by staining with acetylcholine esterase (AChE) and nicotinamide adenine dinucleotide phosphate diaphorase (NADPH-d), respectively. AChE and NADPH-d activity was demonstrated in neuronal cell bodies and nerve fibers in the chicken ileum. The positive neurons showed irregular or polygonal shape and were mainly present sporadic or clumped in the myenteric and submucosal plexus. The positive nerve fibers frequently surrounded the ileac blood vessels. They were abundantly present in myenteric and submucosal plexus of the ileum forming a network. Some positive nerve fibers traversed the submucosa into the lamina propria mucosae. Fine nerve fibers were found to penetrate into intestinal villi underneath the epithelium. Extensive networks of more intensely staining AChE positive nerve fibers were present in the mucosa as compared to that of NADPH-d positive fibers. Ganglia density of submucosal plexus was markedly higher than that of myenteric plexus, whereas neurons per ganglion and the number of neurons per mm2and the size of neurons of submucosal plexus were shorter than that of myenteric plexus. In addition, the number of AChE positive neurons was more than that of NADPH-d positive neurons. We concluded that the chicken ileum is characterized by abundance of nerve structures which may play a significant functional role in ileum of the chicken.