The Significance Of The Expression And Distribution Of Axon Guidance Factors Netrin-1 In Hirschsprung's Disease

ObjectiveHirschsprung's disease is a congenital disorder and occurs in 1:5,000 live births and is characterized by an absence of enteric neurons in terminal regions of the gut. The clinical characteristic of the disease is functional chronic intestinal obstruction, caused by the localized inability of the gut to transmit a motile relaxation and contraction (peristaltic) wave; this section of the bowel is typically contracted and devoid of contents. Current research has proven that Hirschsprung's disease is an enteric nervous system (Enteric nervous system, ENS) developmental disorders which is due to neural crest-derived cells been abnormal in the process of migration, differentiation along the intestinal wall.The enteric nervous system (ENS) is the part of the PNS that operates largely independently of the CNS to coordinate the complex behaviours of the gut. The ENS monitors the state of the lumen and wall of the gut and responds appropriately, activating intrinsic reflexes that generate mixing and propulsive peristaltic movements and change the blood flow and secretions of water and electrolytes. The majority of the ENS derives from migratory vagal neural crest cells originating from the neural tube at the levels of somites 1-7, with different subsets of vagal neural crest contributing to different gut regions. A small proportion of the foregut ENS also derives from migratory anterior trunk neural crest cells of the posterior vagal region. The whole process starts from the 6th week and complete at the 12th week. Netrin-1 is an important member of the Netrins family. Netrin-1 as an axon guidance factor plays an important role in guiding neuritis growth and the migration of gut neural crest cells. At present studying on Netrin-1 in the enteric nervous system focus on aspects of embryology of animals, but expression and distribution of netrin-1 in human intestinal tract has not yet reported. The purpose of this study is to observe distribution and expression of Netrin-1 in normal bowel segment and cramps bowel segment of HD and explore its relationship with the pathogenesis of HD.Methods1. Case specimensCollected 36 cases of paraffin specimens in children with HD at Department of Pathology of our hospital from January 2008 to October 2,009.32 cases were males and 4 females in 1 month to 5 years old age range. These children are sporadic, non-genetic family history of HD.2. Experimental reagents(1) Polyclonal antibodies Netrin-1(H-104:sc-20786) were purchased from Santa Cruz Biotechnology (US).(2) SP test kit and diaminobenzidine(DAB) showing color reagent were Purchased from Beijing Zhong Shan Golden Bridge Co., Ltd..3. Experimental methodswe tested immune response and the distribution of 36 cases of HD in children by immunohistochemistry using anti-Netrin-1 polyclonal antibody.36 cases of children with normal bowel segment were set as normal control group;cramps bowel segment for experimental group. SSPS13.0 for windows used for statistical analysis. Student t test was applied to analyze the significant differents of netrin-1 expression in muscular and mucous layes in control and experimental group.ResultsIn the control group (normal bowel segment), netrin-1 immunoreactivity in the intestinal muscular layers (longitudinal muscle, circular muscles), myenteric ganglias and mucosal layer was very strong,however, and netrin-1 immunoreactivity in experimental group (cramps bowel segment)was also very strong.When the expression of netrin-1 in muscular and mucous layers were compared, no significant defferents were observed between the two group statistically. (ρ>0.05)ConclusionIn this study, the fact that there's no statistically significant difference between the expression of netrin-1 in muscle layer and mucosal layer of normal bowel segment and spasm bowel segment with Hirschsprung'disease may indicate that netrin-1 have no significant correlation with HD.