Expressions of glial cell line-derived neurotrophic factor and its tyrosine kinase receptor RET in the terminal rectum of fetal rats with congenital anorectal malformations at different gestational ages

Abstract

Objective To explore the expressions and distributions of glial cell line-derived neurotrophic factor (GDNF) and itstyrosine kinase receptor RET in the terminal rectums of fetal rats with congenital anorectal malformations (ARM) at different gestationalage, and to explore their effects on the enteric nervous system in the terminal rectum of ARM fetal rats. Methods Thirty-five SD pregnancy rats were divided into a saline group (n=10) and an ethylenethiourea experiment group (n=25) by simple randomized study.The fetal rats were removed from the pregnant rats at the gestational 16 d, 18 d and 20 d. The fetal rats were divided into the saline control group, the ethylenethiourea control group (fetal rats without ARM) and the ethylenethiourea malformation group (ARM fetal rats) by the naked eye and dissecting microscope.HE staining was used to observe the morphology and the intestinal ganglion cells in the terminal rectum were counted.The immunohistochemical staining and Western blot methods were used to observe the distributions of GDNF and RET in the rectum at the gestational 16 d, 18 d and 20 d. The quantitative real-time polymerase chain reaction (qRT-PCR) was used to detect the expression of GDNF mRNA in the fetal rats in the terminal rectum at the gestational 16 d, 18 d and 20 d. Results HE staining: the development of anorectal terminal in 3 groups of fetal rats was unclear at the gestational 16 d. A small amount of scattered nerve plexuses were observed in the muscular layer.The nuclei were small and sparse.The axons and cytoplasms were less.The serosal layer, muscular layer, submucosa, mucosal layer and glands in the terminal rectum were gradually clear in the saline control group and the ethylenethiourea control group at the gestational 18 d and 20 d. The intermuscular submucosal nerve plexuses increased gra-dually (11.400±3.134 and 11.200±3.425 at the gestational 18 d; 66.100±4.954 and 67.600±5.481 at the gestational 20 d). While, the layer was unclear in the ethylenethiourea malformation group and the nerve plexus was less (7.800±1.989 at the gestational 18 d, and 25.200±3.048 at the gestational 20 d), and the difference was statistically significant compared with 2 control groups (F=7.591, 271.833, all P 0.05); the expressions of GDNF and RET protein were 103.624±27.533 and 105.184±19.634 at the gestational 18 d; 151.496±33.622 and 150.738±21.423 at the gestational 20 d in 2 control groups.Compared with the ethylenethiourea malformation group (79.169±11.697 at the gestational 18 d; 94.873±11.309 at the gestational 20 d), and the difference were statistically significant (all P<0.05). Conclusions The expressions of GDNF and its tyrosine kinase receptor RET had a certain temporal correlation in the terminal rectum of normal fetal rats at different gestational ages and ARM.Moreover, the abnormal expressions of GDNF and its tyrosine kinase receptor RET in the distal rectum of ARM fetal rats can affect the development of enteric nervous system. Key words: Glial cell line-derived neurotrophic factor; RET; Signal pathway; Enteric nervous system; Congenital anorectal malformation