Comparative Study Of Enteric Nervous System Development And Regulation In Normal And Anorectal Malformations

IntroductionAnorectal malformations(ARMs)are one of the most common digestive disorders, affecting 1/5000 to 1/1500 live births,one kind of malformations supervised by World Health Organization.In spite of the recent advances in operative techniques,the long-term prognosis in intermediate and high type ARMs remains poor.Some patients with ARMs continued to suffer from postoperative anal dysfunctions.The anorectal dysfunction was caused by the dysplasia of the neuromuscular in the pelvic floor. Undoubtedly,the intestinal motility is the focal point of the organism of continence. The research of the development of the enteric nervous system,intestinal muscle and nerve-muscle contact and the possible control mechanisms in ARMs has been necessary.In previous studies,it has been shown that in the middle and high ARMs,sacral cord and the enteric nervous system of the rectal end have changed differently,but so far the course of their embryonic development and detailed pathological changes are unclear.With the progress of molecular biology,the embryogenesis of the enteric nervous system has been illuminated initially.Vagal and sacral neural crest cells give rise to enteric neurons and glial cells throughout the entire gastrointestinal tract.In ARMs, after the defect appears,there has been a question that if the abnormity of the enteric nervous system accompanied the ARMs.The etiopathogenisis of ARMs continues poorly understood and controversial.In current study,it has been only demonstrated that ARMs resulted from hereditary factors and environmental factors in the development of embryogenesis.During the research of epidemiology and animal experiments,it is suggested that the hereditary factors play a key role in the development of ARMs.Recently,it was found that Shh,Hox,Fgf10 and Wnt5a play crucial roles during the development of the termination of digestive tract.Once the foresaid genes dysfunction,there may be ARMs,such as anal atresia,preternatural anus et al.In recent years research has shown that the highly conserved Wnt signaling protein family in embryonic development play an important role in signaling through different molecular interactions,Wnt proteins trigger the regulation of cell growth,migration, differentiation and development,and many other complex signaling cascade reaction, Wnt5a is an important parterner in this family.Most of its researches involving human subjects are common in all systems tumor,at the same time it play an important role in the embryogenesis of intestinal tract,but the its regulation of intestinal smooth muscle and enteric nervous system during embryonic development have not been reported, especially it is not clear for the mechanism of the intestinal neuromuscular dysplasia in ARMs.In this paper,we utilize the Wistar rat animal model of ARMs induced by ETU (ethylenethiourea).We used PGP9.5(protein gene product 9.5)and SYP (synaptophysin)and NOs(nitric oxide synthases)for evaluating the enteric nervous system and neuromuscular development of intestinal tract,at the same time we analyzed the expression of Wnt5a and its receptor Frizzled-1,TCF4 protein and mRNA in the rectum of normal and ARMs to explore the possible regulatory mechanism of enteric nervous system and gastro-intestinal smooth muscle of the embryonic period,which is the important role in participate in defecation.In addition, we used the clinical specimens in children with ARMs to explore the Wnt5a protein expression.MaterialsAnimals:Wistar rats(250-300g)were provided by Medical Animal Center in Shengjing Hospital of China Medical University.Clinical specimens:the case group:20 ARMs infants operated in our department. Tissues were removed from terminal pouch of rectum and stored immediately in fluid nitrogen.In the control group,tissues were obtained at 1 cm superior to dentate line in acquired anal fistula and rectal traumas infants.Reagents:①The specific antibody to PGP9.5 was purchased from Abcam Corporation.②The specific antibodies to SYP,Nos,Wnt5a,Frizzled-1,TCF4 were purchased from Santa Cruz Corporation.③The UltraSensitiveTMSP kits for immuhistochmistry were purchased from MaiXin biological limited company,FuJian Province.Two-step immunohistochemical goat detection kit was purchased from Beijing Zhong Shan Golden Bridge Company;④Ethylenethiourea,ETU was purchased from Sigma-Aldrich Inc.,Germany;⑤Reverse transcriptase kit was purchased from Takara Bio-engineering Co.,Ltd;⑥Total protein extraction kit was purchased from Seitz Corporation.MethodsTime-mated pregnant Wistar rats were gavage-fed a single dose of either 125 mg/kg of 1%ETU(2-Imidazolidinethione,98%;Aldrich Chemical Co,Inc,Germany) or an equal dose of saline on gestational day 10(E0—sperm in vaginal smear after overnight mating).The embryos were harvested via cesarean section on E15 to E21. The embryos were fixed in 4%paraformaldehyde/0.1 M phosphate buffered saline (PBS)for 12 to 24 hours depending on their size.Then the embryos from each age group were dehydrated,embedded in paraffin and sectioned serially transversely,and sagittally at 4μm thickness.①Immunohistochemistry and fluorescence staining of PGP9.5 and SYP,NOs(embryonic development ENS markers)were to observe normal and ARMs rat embryonic development of the ENS.②Immunohistochemical staining of Wnt5a,Frizzled-1 and TCF4 were compared to roughly to position at the intestinal wall.Another part of rectum samples of rats were drawn by microscope,we evaluated the expression of Wnt5a,Frizzled-1 and TCF4 mRNA by RT-PCR and we evaluated the expression of PGP9.5,SYP,NOs,Wnt5a,Frizzeled-1,TCF4 prtein by the western blot quantitative analysis.G:BOX SYNGENE photographed gel analysis system and Kodark Digital Science 1.0 DNA analysis software to carry out semi-quantitative analysis.T-test was used to compare the differences of foresaid genes expression levels between the normal and ARMs groups,P＜0.05 indicates that the difference has statistical significance.③Immunohistochemical staining and western blot analysis of Wnt5a were used to compare the expression level in the the case group and the control group.Results1.The embryonic development of the ENS in normal and ARMs rat embryos①PGP9.5 labeled neurons in the post-intestinal migration and development:Immunohistochemical results showed that in normal control group,at E16:neural crest cells already colonized in the whole intestine and scattered positive cells were seen in the rectum,when the circular muscles has just begun to differentiate;From E16 to E18,PGP9.5-positive cells appeared in the myenteric plexus sites poradically;At E17-18,the terminal of the longitudinal intestinal muscle fiber distribution is still sparse and there were rare PGP9.5 positive cells in the submucosa;At E20:it can be observed that the yellowish-brown mass of PGP9.5-positive ganglion cells,showing cluster-like.The positive cells also can be seen in the submucosa.At E21,it become more obvious.While at the same sections observed in the same period in the intestinal wall,the development of ENS grew earlierly.In ARMs Group,at E16-17,neural crest cells at the terminal of hindgut malformation group could also be seen and there was no significant difference between the two groups,but there was no positive cells around rectal urethral fistula;At E18-19,more positive cells were still visible near the rectum terminus,but the distribution compared with the normal group is sparse,yet no positive cells were observed around the fistula;At E20-21,compared with the normal and upper colon,the density of positive cells reduced in the rectal terminus,and no obvious clusters of nerve cells were seen.The development of ENS in ARMs lags behind the normal group.②The development of nitrergic neurons marked by NOs:The immunofluorescence results showed the development of nitrergic neurons accompanied neural crest cell migration during embrygenesis,but it was a little later than the emergence of PGP9.5.They appeared at around E17 in the rectum.At E18,the clear positive neurons appeared between the muscles of the rectum.The cytoplasm has full of obvious green fluorescence.Before birth the development of positive neurons have become more sophisticated.But in ARMs group compared with the normal group, the growth of nitrergic neurons have lagged.The older,the greater of the difference between the two groups.③Immunofluorescence results of SYP:In normal control group,only scattered fluorescent display were observed in the top of the colon wall at E16,but they are very weak;From E18,strong green fluorescence can be seen clearly within in the intestinal myenteric position.It is straight into a cord-like and was more clear in the upper colon than the rectum.To E20,as well as before birth,fluorescence intensity gradually increased.But in ARMs Group,at E16-17,no fluorescence were shown in the majority of colorectal.Only a very weak visible fluorescence could be found in the upper colon.At E18,the development of SYP in the upper colon became more sophisticated,but in the blind-side near the rectum green fluorescence was still visible.Compared with the upper segment of normal colon and normal group,the intensity of fluorescence in ARMs decreased,there were no fluorescence around the fistula.④The western blot analysis of PGP9.5,SYP and NOsThe results showed that the protein of the above-mentioned factor had significant differences between the two groups on E19-21.2.Expression of Wnt 5a and other development-related factors in normal and ARMs rat embryos①Immunohistochemical staining results of Wnt5a,Frizzled-land TCF4:in the normal group from the E16～E21,the expression of the above-mentioned factors were observed in the myenteric plexus,the mucosa and intestinal smooth muscle of the rectum,but the intensity of expression were different.As the development of the rectum,the expression become more increased.In ARMs Group,at E16 there were no positive cells.From E17-E21,the positive cells gradually becoming apparent,with the normal gestational age of the rectum.But compared to the same region of the normal group,the expression decreased,no positive cells appeared around the fistula.②The western blot results of Wnt5a,Frizzled-1 and TCF4 showed on E17,there was no significant difference between the two groups;on E19-E21,the expression levels of Wnt5a,Frizzled-1 and TCF4 were higher in the normal group than in ARMs with significant difference(p＜0.05).③RT-PCR results of Wnt5a,Frizzled-1 and TCF4 mRNA showed that there have been a significant difference between the two groups.3.Expression of Wnt5a in normal and ARMsIn the anorectal tissues of the human of the normal case,the expression of wnt5a was mainly located in the cytoplasm of the myenteric plexus by immunohistochemistry. But in the ARMs group,we did not found positive cells in the myenteric plexus of the the blind part of the rectum espesially in high ARMs.There was no significant difference of wnt5a protein expression level between high and intermediate ARMs by western blot analysis(P=0.33).However,the expression levels of wnt5a protein in high and intermediate ARMs were significantly lower than that in low group(P＜0.05). There was no significant difference of wnt5a protein expression level between the infected recto-vestibular fistula and the control group(P=0.67).Conclusions1.In normal and ARMs rat embryos,on E16,neural crest cells migrate to the rectal area,but the development of rectal terminus is worse.With the further development of rat embryos,the enteric nervous system in rectal region improved gradually,plexus-like structure has gradually formed,and postnatal development became more perfect.In ARMs group,on E16,neural crest cells could also be seen in the terminal of hindgut,there was no significant difference between the two groups. From E18,ARMs group compared with the normal group,the development of ENS has lagged;the older,the greater the difference between the two groups.2.The developmental course of the nitrergic neurons is accompanied by neural crest cell migration and development,but it is some later.It is suggested that NO may play an important role during the development of enteric nervous system.In ARMs group,there was significant difference of synaptophysin and NOs expression between the two groups in the rectal terminal region,which may lead to change to intestinal motility.3.E16～E21,compared with in the normal group,it is in ARMs group,the expression of Wnt5a,its receptor Frizzled-1 and TCF4 which is a classical key factor of Wnt pathways in intestinal smooth muscle and nervous system became significantly weakened.It is suggested that Wnt 5a,which may play a role by the classical pathway and may be related with the developmental regulation of enteric nervous system and smooth muscle of intestinal tract in ARMs.4.Wnt 5a may play a role in the developmental regulation of the ENS in ARMs.