Interstitial Cells Of Cajal In Hirschsprung's Disease

Aganglionic megacolon, i.e. Hirschsprung's disease (HD), is a functional intestinal obstruction with an incidence of 1/3000-1/5000 live births. This disorder is characterized by severe constipation due to the absence of enteric ganglia along a variable length of the intestine. Varying lengths of the distal colon are unable to relax, causing functional colonic obstruction over time. Symptoms range from neonatal intestinal obstruction to chronic progressive constipation in older children. With regard to the treatment of aganglionic gut conditions, surgical intervention is frequently the only option, whereas surgical therapy does not always lead to a complete recovery and restoration of all bowel functions, which followed by some complications such as constipation, fecal incontinence and most serious enterocolitis, colonic rupture and psychic problem. Some researchers elucidated the possibility of intracolonic grafting of neural stem cells(NSCs) as a therapeutic methed for HD. Although the grafted NSCs can survive, differentiate into neuronal phenotypes and partly improve denervated colon motility recovery in the aganglionic colon wall after transplantation. The results were not so good, indicating that there might be some other factors participating in the development of gastrointestinal motility disorders in HD. Recently more and more attention has been paid to the studies associated with interstitial cells of Cajal.Interstitial cells of Cajal (ICCs) was first described in 1893 by the great Spanish neuroanatomist, Santiago Ramon y Cajal. ICCs were located between gastrointestinal smooth muscle cells and nerve endings. After more than a century studies of morphology and physiology, investigators have been certain that ICCs are neither neurons nor glial cells, but not smooth muscle cells. They form cellular network in gastrointestinal tract and play a very important role in the regulation of the gastrointestinal motility by generating and propagating spontaneous electric slow-vave activity and mediating excitatory and inhibitory neurotransmission. Many studies show that decreased numbers or disrupted networks of ICCs were associated with gastrointestinal motility disorders, including Hirschsprung's disease. To study the relativity between abnormalities of ICCs and HD will help understanding the pathogenesis of HD, providing new base for cell transplantation research.Based on the above concept, we addressed this issue to study the relationship between interstitial cells of Cajal and Hirschsprung's disease deeply. This study can be divided into three parts:Part one: Isolation and culture of interstitial cells of Cajal from rat colon in vitroAlthough there are many researches on ICC now, the procedure in isolation and culture of ICC still has many difficulties that should be studied and discussed. In order to identify ICCs more accurately from cell morphology, we make much efforts in studying the isolation, culture ICCs from rat colon in vitro. As we know, the quantity of ICCs in gastrointestinal tract is very small. They are located between smooth muscle cells and nerve fibers, embeded in dense connective tissue matrix. It is very difficult to isolate ICCs from rat colon. After trying so many times, using Collagenase and pancreatin digestion and ficoll density centrifugation, we successfully isolated and cultured ICCs from rat colon in vitro. The antibody of c-kit (CD117) was used to identify the cultured ICCs. The colon tissue of Wistar rat was dissected. Collagenase and pancreatin were used to isolate ICCs. The cell suspension was resuspended in M199 medium containing stem cell factor (stem cell factor, SCF). After 24 hours, we changed the medium for the first time. Then the medium was changed every other day. After 3 days, cultured ICCs exhibited a few axis-cylinders and longer axis-cylinders were observed to form synapse each other after 5 days. More widespread connections formed within 7 days in vitro. The change of its morphologic character were obvious within 7days. Flourescent staining with c-kit antibody confirmed that the cultured ICCs was successful.Part two: The distribution pattern of interstitial cells of Cajal in children colon.Normal colon specimens were obtained from seven children (age from 5 months to 26 months, mean age 11.6 months,5 boys and 2 girls) at the time of bladder augmentation. All specimens were obtained from the same part of a relevent region immediately after resection. Frozen serial sections (10μm) were cut on a cryostat and mounted onto slides coated with 0.1% poly-L-lysine and stored at 4℃. The distribution of interstitial cells of Cajal was visualized by immunohistochemistrical method using monoclonal mouse anti-human c-kit (CD117) antibody. c-Kit positive ICCs formed a dense network surrounding the myenteric plexus (IC-MY), in submucosal border (IC-SM) and smooth muscle layer (IC-IM). IC-MY formed a dense network encasing the myenteric plexus. IC-IM appeared as long, thin, bipolar cells with one or two processes connecting with each other. NOS positive neurons were evaluated by nNOS fluorescent staining on whole-mount preparations and frozen serial sections. A great number of NOS positive neurons and nerves were found within the circular muscle layer and myenteric plexus of the colon. Double labelling of nNOS and c-kit immunohistochemistry on frozen serial sections revealed a close distribution between NOS positive nerves and ICCs network surrounding myenteric plexus, which suggested that both NOS positive nerves and ICCs contribute to normal gastrointestinal tract motility.Part three: Altered distribution of intersitial cells of Cajal in Hirschsprung's disease.Full thickness colonic specimens were obtained from five children with total colonic aganglionosis (TCA), sixteen with recto-sigmoid HD and seven controls. Frozen serial sections (10μm) were cut on a cryostat and mounted onto slides coated with 0.1% poly-L-lysine and stored at 4℃. The distribution of interstitial cells of Cajal and NOS positive neurons were visualized by c-kit (CD117) and nNOS fluorescent staining on frozen serial sections respectively. Morphometric analysis was performed to determine the distribution and density of c-Kit -positive ICCs in controls and patients. In the aganglionic colon of HD, ICCs located in the three regions (IC-SM, IC-IM, IC-MY) were markedly decreased in number. NOS positive nerves were aslo reduced in number. Expression of c-kit and nNOS mRNA were detected by reverse transcriptase-polymerase chain reaction (RT-PCR), while expression of c-kit and nNOs protein were detected by Western blot analysis. The results show that c-kit mRNA and protein expression significantly declined in the HD group. Decreased expression of nNOS mRNA and protein were demonstrated in the HD group compared with the control group. The declined expression of c-kit gene and nNOS gene were associated with HD.Conclusions1. We have established the method to isolate and culture interstitial cells of Cajal from the Wistar rat colon in vitro. And our study offer the base for investigating the biologic property of ICCs.2. The distribution pattern of interstitial Cajal cells, NOS-positive neurons and smooth muscle cells in the children colon is similar to that of adult. Each of them play an important role in conrolling gastrointestinal tract motility and physiological function. NOS-positve neurons in human intestinal myenteric plexus might have some effects on IC-MY.3. We demonstrated the distribution and density of different subtypes of ICCs within the colon of recto-sigmoid HD and TCA patients and that each subtype of ICCs was decreased in number or was even lacking. The lack or reduction of ICCs in TCA and HD patients may lead to defective generation of pacemaker activity, thus contributing, together with the lack of neurons, to the motility dysfunction present in HD.