The Expression Of NGF And NGFR In Distal Rectum In Fetal Rats With Anorectal Malformations

Objective:To explore the development of the enteric nervous system(ENS) by detecting the expression of nerve growth factor(NGF) and growth factor receptor(NGFR) TrkA and p75NTR in distal rectum in fetal rats with anorectal malformations(ARM).To study the mechanism of defecation functional perturbation in patients with ARM.Methods:16adult pregnant SD rats were divided into ARM group (n=10) and normal control group (n=6).The intragastric administration of ethylenethiourea(ETU) was performed on fetal rats to create anorectal malformation model while distilled water to normal control group on the11th and13th day of gestation.On the20th day after pregnancy, the cesarean section fetus were obtained for morphological observations and abnormal statistics.Using hematoxylin-eosin staining(HE) to detect the perineum of sagittal gross specimen.To count the number of submucosal and myenteric plexus of distal rectum in fetal rats:Using immunohistochemistry(IHC) to study the expression and distribution of NGF、TrkA and p75NTR of distal rectum in fetal rats:Using real time-Polymerase Chain Reaction(RT-PCR) to detect the expression levels of TrkA and p75NTR of distal rectum in fetal rats.To take the gene amplification cDNA fragment for agarose gel electrophoresis.Results:①Morphological observations and abnormal statistics:Compared with normal control group whose survival rate was100%and no rectal abnormal openings.the ARM group’s survival rate was80.7%. ARM was98.7%. short-tailed deformity was5.1%, no tail deformity was94.8%. varus feet was6.4%. spina bifida was6.4%.Malformations may-be single may also be complex.②HE staining:The ARM fetal rectums which had no structure of the normal anus was hyperplastic squamous epithelial and thickened muscular layer and with no intestinal glands distribution.In term of the number of submucosal and myenteric plexus.they presented remarkably statistical differences via comparison(Normal control group:96.33±18.57:while the induced ARM group:46.40±16.08)(P<0.05):③IHC:The NGF、TrkA、p75NTR were mainly distributed in the fetal rats terminal rectum mucosa and myenteric neurons. NGF was mainly localized in the nucleus. TrkA was in the cytoplasm and p75NTR was in the nucleus and cytoplasm.In the normal control group, the integral optical density(IOD) value of NGF and TrkA.as well as p75NTR were respectively731.75±232.18,262.82±101.72and134.67±67.13while in induced ARM group they were217.97±74.95.113.0±36.47and33.15±11.13respectively.There are significant differences between both groups(P<0.05);④RT-PCR:In term of relative expression of TrkA and p75NTR mRNA.they presented remarkably statistical differences via comparison(normal control group:162.22±104.68and129.78±51.98;while the induced ARM group:78.94±33.43and87.15±25.81)(P<0.05).In agarose gel electrophoresis.the gene amplification cDNA fragment was consistent exactly with primer design.and normal control group electrophoretic bands was brighter than ARM group.Conclusion:The abnormal expression of NGF and NGFR. might have played an important role in the development of the enteric nervous system in fetal rats with ARM and might be one of the reasons that lead defecation functional perturbation in patients with ARM.