Correlation Study On Innervations Distribution In The Rectal End With Anorectal Malformations

Congenital anorectal malformations(ARMs) are one of the most common and severe gastrointestinal anomalies in the neonatal period.The incidence of ARMs ranges from 1 in 1,500 to 5,000 live births.Although morphological anomalies may be reshaped by the surgical treatments,postoperative defecation disorders such as soiling and constipation occur in the part of patients.Recent study reported that there was close correlation between the good bowel movements and enteric nervous system(ENS) in the rectal end.Vitamin A and folic acid play important parts in the biological embryogenesis,generative differentiation and normal homeostasis.Animal experiments proved that vitamin A and folic acid deficiency or over-dose in the duration of pregnancy caused fetal mice anomalies.But there are no reports about the influence of vitamin A and folic acid on the anorectal upgrowth.In addition,the development of ENS may be adjusted by the neurotrophic factors.This study was divided into four parts.In part 1,we would study ENS in the rectal end with different types of ARMs to explore the possible pathological mechanisms of postoperative defecation disorders.In part 2,by comparing fetal mice ARM model induced by ethylenethiourea,we would study the effect of vitamin A on the teratogenesis of ARMs and the ENS upgrowth in the rectal end.In part 3,we would study the effect of folic acid on the teratogenesis of ARMs and the changes of DNA methylation in the rectal end.In part 4,we would study the influence of extrinsic glial cell line-derived neurotrophic factor(GDNF) on the ENS upgrowth in the rectal end, and discuss whether the extrinsic intervention may advance the expression of GDNF gene,which may contribute to ENS dysplasia developing mature.Part 1:Clinical study on the expression of PGP 9.5 and S-100 protein in the rectal end with congenital anorectal malformations Objective:To study ENS in the rectal end with clinical different types of ARMs and explore the possible pathological mechanisms of postoperative defecation disorders.Methods:76 cases of rectal end samples came from the patients with ARMs from January 2001 to December 2005 in Children's Hospital of Fudan University,of which high-type was in 11 cases,intermediate-type in 26 cases,and low-type in 39 cases. The mean fetal age,birth weight and the age acquiring the rectal samples were analysed and the expression of PGP 9.5 and S-100 protein in above samples were visualized by immunohistochemistry.Results:1.There were no significant differences in the mean fetal age and birth weight among the different types of ARMs(P＞0.05).2.Immunohistochemical results:(1) The expression of PGP 9.5 and S-100 protein in the high-type anomalies was obviously lower than that in the intermediate- and low-type anomalies(P＜0.05),but there were no markedly differences between the intermediate- and low-type anomalies (P＞0.05).(2) There were no obvious differences in the age acquiring the rectal samples between the high-type group and the staged-procedure group of the intermediate-type anomalies(P＞0.05),but the expression of PGP 9.5 and S-100 protein in the staged-procedure intermediate-type group was obviously higher than that in the high-type group(PGP 9.5:P=0.0070;S-100:P=0.0041).(3) The expression of PGP 9.5 and S-100 protein in the one-stage procedure group of the intermediate-type anomalies was markedly lower than that in the staged-procedure group(PGP 9.5:P=0.0128;S-100:P=0.0048).(4) There was a significant difference in the age acquiring the rectal samples between the male group and the female group of the low-type anomalies(79.27±58.78 hours vs 6.15±0.29 months,P＜0.0001),but there were no statistical differences in the expression of PGP 9.5 and S-100 protein(P＞0.05).Conclusions:There is a close relation between the developmental extent of the ENS and the abnormal position of the ARMs.The high incidence of postoperative defecation disorders in the high-type anomalies may be related with the poor development of the ENS in the rectal end.The development of the ENS in the rectal end with intermediate-type anomalies may gradually grow mature with the age. Part 2:The effect of vitamin A in the duration of pregnancy on the anorectal malformationsObjective:To study the effect of vitamin A on the teratogenesis of ARMs and the ENS upgrowth in the rectal end by comparing fetal mice ARM model induced by ethylenethiourea.Methods:80 female Sprague-Dawly(SD) mice(200～250g) were randomly divided into 4 groups,namely VAD group(n=20) with vitamin A deficient diet from two weeks before the pregnancy to the pregnant end,high-VA group(n=20) with vitamin A supplementary diet from two weeks before the pregnancy to the pregnant end, normal-VA group(n=20,as negative control group) and ETU group(n=20,as positive control group) with normal diet from two weeks before the pregnancy to the pregnant end.In the tenth day of the pregnancy,ETU group was gived 1%ETU by the stomach tube and the other three groups were gived the equivalent normal saline.In the twentieth day of the pregnancy,all pregnant mice were performed the cesarotomy. The conceptive rate,weight,death rate,abortive rate,mean fetal numbers in each nest, still-birth rate,the incidence of ARMs,fetal weight and nose to stern length were recorded,and the serum concentrations of vitamin A in the pregnant mice and the expression of PGP 9.5 and S-100 protein in the rectal end of the fetal mice were detected by immunohistochemistry.Results:1.The serum concentrations of vitamin A in VAD group which was fed with vitamin A deficient diet for two weeks were obviously lower than that in normal-VA group (P=0.0310) and ETUgroup(P=0.0401).The serum concentrations of vitamin A in high-VA group which was fed with vitamin A supplementary diet for two weeks were evidently higher than that in normal-VA group(P=0.0036) and ETU group(P=0.0026).2.Pregnant mice:(1) The weights of the pregnant mice in normal-VA group and ETU group presented trend growth,and there were no significant differences in the different phases between the two groups(P＞0.05).The weights of the pregnant mice in VAD group and high-VA group also presented trend growth before the tenth day of the pregnancy,but presented downtrend after the tenth day of the pregnancy.The weights in the same phases in VAD group and high-VA group were evidently lower than that in normal-VA group and ETU group(P＜0.01). (2) There were no significant differences in the conceptive rate and death rate among the four groups(P＞0.05).(3) The abortive rate of the pregnant mice in VAD group and high-VA group was 76.9%and 100.0%,respectively,which was significantly higher than that in normal-VA group(P＜0.0001) and ETU group(VAD group vs ETU group,P=0.0009; high-VA group vs ETU group,P＜0.0001).3.Fetal mice:(1) The mean fetal numbers in each nest of VAD group were obviously fewer than that in normal-VA group(P=0.0040) and ETU group(P=0.0053),but there were no significant differences between normal-VA group and ETU group(P＞0.05).(2) The still-birth rate in VAD group,normal-VA group and ETU group was 36.4% (8/22),1.2%(2/169) and 5.0%(7/140),respectively.There were significant differences in the still-birth rate among the groups(P＜0.05).(3) The fetal weight and nose to stern length in VAD group were evidently lower than that in ETU group and normal-VA group(P＜0.0001),but there were no significant differences between ETU group and normal-VA group(P＞0.05).4.The incidence of ARMs in VAD group and ETU group was 71.4%(10/14) and 45.9%(61/133),respectively.There were no statistical differences both in the incidence of ARMs and in the concomitant tail anomalies between the two groups (P＞0.05).There were no fetal mice with ARMs in normal-VA group.5.Immunohistochemical results:(1) In the rectal end of fetal mice with normal anus,the expression of PGP 9.5 and S-100 protein in VAD group was evidently lower than that in ETU group(PGP 9.5:P =0.0156;S-100:P=0.0105) and normal-VA group(PGP 9.5:P=0.0091;S-100:P= 0.0024),but there were no significant differences between ETU group and normal-VA group(P＞0.05).(2) In the rectal end of fetal mice with ARMs,the expression of PGP 9.5 and S-100 protein in VAD group was evidently lower than that in ETU group(P＜0.0001).And the expression of the two antibodies in the rectal end of fetal mice with ARMs was obviously lower than that in the rectal end of fetal mice with normal anus, respectively(VAD group:P values of PGP 9.5 and S-100 were both less than 0.0001; ETU group:P values of PGP 9.5 and S-100 were 0.0203 and 0.0122,respectively).Conclusions:The vitamin A deficiency in the duration of pregnancy may result in the teratogenesis of ARMs,and the developmental extent of the ENS in the rectal end of the fetal mice may be related with the serum concentrations of the maternal vitamin A.Part 3:The effect of folic acid in the duration of pregnancy on the anorectal malformationsObjective:To study the effect of folic acid on the teratogenesis of ARMs and the changes of DNA methylation in the rectal end.Methods:80 female SD mice(200～250g) were randomly divided into 4 groups, namely FD group(n=20) with folic acid deficient diet from two weeks before the pregnancy to the pregnant end,high-FA group(n=20) with high-dose folic acid diet from two weeks before the pregnancy to the pregnant end,normal-FA group(n=20) and control group(n=20) with normal diet from two weeks before the pregnancy to the pregnant end.In the tenth day of the pregnancy,FD group,high-FA group and normal-FA group were gived 1%ETU by the stomach tube and the control group was gived the equivalent normal saline.In the twentieth day of the pregnancy,all pregnant mice were performed the cesarotomy.The conceptive rate,weight,death rate,abortive rate,mean fetal numbers in each nest,still-birth rate,the incidence of ARMs,fetal weight and nose to stern length were recorded,and the serum concentrations of folic acid and the expression of GDNF methylation in the rectal end of the fetal mice was evaluated by PCR.Results:1.The serum concentrations of folic acid in FD group which was fed with folic acid deficient diet for two weeks were obviously lower than that in normal-FA group (P=0.0204) and control group(P=0.0080).The serum concentrations of folic acid in high-FA group which was fed with high-dose folic acid diet for two weeks were evidently higher than that in normal-FA group(P=0.0242) and control group (P=0.0405).2.Pregnant mice:(1) The weights of the pregnant mice in each group presented trend growth.The weight increases on the tenth day and the twentieth day of the pregnancy in FD group were obviously lower than that in the other three groups(P＜0.05),but there were no significant differences in the different phases among the other three groups(P＞0.05).(2) There were no significant differences in the conceptive rate between FD group and high-FA group(P＞0.05),but the conceptive rate in the two groups was lower than that in normal-FA group(P=0.0392) and control group(P=0.0124);there were no statistical differences among high-FA group,normal-FA group and control group (P＞0.05).(3) There were no significant differences in the death rate and abortive rate among the four groups(P＞0.05).3.Fetal mice:(1) The mean fetal numbers in each nest of FD group were obviously fewer than that in high-FA group(P=0.0002),normal-FA group(P=0.0043) and control group (P=0.0032),but there were no significant differences among high-FA group, normal-FA group and control group(P＞0.05).(2) The still-birth rate in FD group,high-FA group,normal-FA group and control group was 7.7%(6/78),3.8%(6/158),5.0%(7/140) and 1.2%(2/169),respectively. There were significant differences in the still-birth rate between FD group and control group(P=0.0071) and between normal-FA group and control group(P=0.0472),but there were no statistical differences among the other groups(P＞0.05).(3) The fetal weight and nose to stern length in FD group and high-FA group were evidently lower than that in normal-FA group and control group(P＜0.0001),but there were no significant differences between FD group and high-FA group(P＞0.05) and between normal-FA group and control group(P＞0.05).4.The incidence of ARMs in FD group,high-FA group and normal-FA group was 51.4%,42.8%and 45.9%,respectively.There were no statistical differences both in the incidence of ARMs and in the concomitant tail anomalies among the three groups (P＞0.05).There were no fetal mice with ARMs in the control group.5.Results of GDNF methylation:(1) In the rectal end of the fetal mice with normal anus,the expression of GDNF methylation in FD group was evidently lower than that in high-FA group(P＜0.0001), normal-FA group(P=0.0003) and control group(P=0.0004),but there were no significant differences among high-FA group,normal-FA group and control group (P＞0.05).(2) In the rectal end of the fetal mice with ARMs:①The expression of GDNF methylation in FD group was evidently lower than that in high-FA group and normal-FA group(P＜0.0001),and the expression in normal-FA group was also obviously lower than that in high-FA group(P＜0.0001).②The expression of GDNF methylation in the rectal end of the fetal mice with ARMs in FD group and normal-FA group was obviously lower than that in the rectal end of fetal mice with normal anus,respectively(FD group:P=0.0335;normal-FA group: P=0.0267),but the expression in the rectal end of the fetal mice with ARMs in high-FA group was evidently higher than that in the rectal end of the fetal mice with normal anus(P=0.0004).③In comparison with the control group,the expression of GDNF methylation in the rectal end of the fetal mice with ARMs in normal-FA group was lower(P=0.0190) and in high-FA group was still higher(P＜0.0001).Conclusions:Maternal folic acid deficiency may cause the shortage of methyl groups, which made the genome in the rectal end of the fetal mice in low methylation conditions.The developmental extent of the ENS in the rectal end of the fetal mice with ARMs may be related with the expression of the tissue methylation.Part 4:The effect of extrinsic neurotrophic factor in the duration of pregnancy on the enteric nervous system in the rectal end with anorectal malformationsObjective:To study the influence of the extrinsic glial cell line-derived neurotrophic factor(GDNF) on the ENS upgrowth in the rectal end,and discuss whether the extrinsic intervention may advance the expression of GDNF gene,which may contribute to ENS dysplasia developing mature.Methods:30 female SD mice(200～250g) which were ensured embryonate were randomly divided into 3 groups,namely ETU group,ETU+GDNF group and control group,and each group contained 10 mice.In the tenth day of the pregnancy,ETU group and ETU+GDNF group were gived 1%ETU by the stomach tube and the control group was gived the equivalent normal saline.In the eleventh day of the pregnancy,ETU+GDNF group was injected with 20μg GDNF(dissolved in 0.2 ml normal saline) by caudal vein,and ETU group and control group were injected with the equivalent normal saline.In the twentieth day of the pregnancy,all pregnant mice were performed the cesarotomy.The death numbers of the pregnant mice,mean fetal numbers in each nest,still-birth rate,the incidence of ARMs and fetal weight and nose to stern length were recorded,and the expression of GDNF in the rectal end of the fetal mice were detected by immunohistochemistry and western-blotting.Results:1.All the pregnant mice were not dead or abortive,and there were no marked differences in the mean fetal numbers in each nest,still-birth rate,and fetal weight and nose to stem length in each group(P＞0.05).2.There were no significant differences in the incidence of ARMs between ETU group and ETU+GDNF group(51.4%vs 52.5%,P＞0.05),and there were no fetal mice with ARMs in the control group.2.Results of immunohistochemistry and western-blotting:(1) In the rectal end of the fetal mice with normal anus,there were no significant differences in the expression of GDNF among ETU group,ETU+GDNF group and control group(P＞0.05).(2) In the rectal end of the fetal mice with ARMs:①The expression of GDNF in ETU+GDNF group was evidently higher than that in ETU group(P=0.0366),and the expression in the rectal end of the fetal mice with ARMs in ETU group and ETU+GDNF group was obviously lower than that in the rectal end of fetal mice with normal anus,respectively(ETU group:P=0.0012; ETU+GDNF group:P=0.0285).Furthermore,the expression of GDNF in the rectal end of the fetal mice with ARMs in ETU group and ETU+GDNF group was evidently lower than that in the control group(ETU group vs control group,P＜0.0001; ETU+GDNF group vs control group,P=0.0265).②The expression of GDNF by western-blotting in ETU+GDNF group was significantly higher than that in ETU group(P＜0.0001).But the expression in ETU+GDNF group is still obviously lower than that in the control group(P=0.0076).Conclusions:The extrinsic GDNF may not block the teratogenesis of ARMs induced by ethylenethiourea,but may advance the expression of GDNF gene in the rectal end and promote the development of the dysplastic ENS.