The Significance Of The Expression Of GDNF And Ret In Distal Rectum In Fetal Rats With Anorectal Malformations

Objective:To explore the development of the enteric nervous system by detecting the expressionof GDNF and its receptor RET in distal rectum fetal rats with anorectalmalformations(ARM). To provide theoretical basis for the treatment of postoperativebowel dysfunction in children.Methods:40adult pregnant SD rats were divided into ARM group (n=30) and normal controlgroup(n=10).The intragastric administration of ethylenethiourea (ETU) was performed onfetal rats to create anorectal malformation model while distilled water to normal controlgroup on the10thday of gestation. In day16,18and20after pregnancy, the cesareansection fetus were obtained for morphological observations and abnormal statistics.Thefetal rats were divided into saline control group, ETU control group (fetal rats withoutARM), ETU malformation group (fetal rats with ARM). Using hematoxylin-eosin staining(HE) to detect the distal rectum in fetal rats then count the number of submucosal andmyenteric plexus of distal rectum of the3groups in day16,18and20. Usingimmunohistochemistry (IHC) and western blot to study the protein expression anddistribution of GDNF and RET of distal rectum in fetal rats; Using quantitive realtime-polymerase chain reaction(qRT-PCR) to detect the mRNA expression levels ofGDNF and RET of distal rectum in fetal rats.Results:①Morphological observations: the control group was given birth to126fetal rats withthe survival rate of100%, no rectal abnormal openings, the ARM group was given birth to208fetal rats with the survival rate of100%, and the rate of ARM was59.135%, it includesno anal deformity, no tail or short tail, meningocele or sacral meningocele, limb anomalies,gastroschisis deformity and so on.②Comparison of tissue morphological change and anus rectum terminal plexus infetal rats of the3groups: Saline control group and ETU control group grow with normalanus while there was no opening at the end of the distal rectum of the ARM fetal rats, the end of the rectum had thickened muscular layer and covered by hyperplastic squamousepithelial. No statistically significant difference in rectum terminal nerve plexus ofbetween saline control group and ETU control group in day16,18and20(P>0.05). Nostatistically significant difference with three groups of fetal rats in day16(P>0.05)，there issignificant difference in saline control group, ETU control group compared with ETUmalformation group in day18were statistical significance(11.400±3.134vs7.800±1.989,P<0.05;11.200±3.425vs7.800±1.989, P<0.05). There were significant difference in day20when3groups were compared (66.100±4.954vs25.200±3.048, P<0.01;67.600±5.481vs25.200±3.048, P<0.01). No statistically significant between saline control group andETU control group in day16,18and20(P<0.01). Nerve plexus of rectum terminal wasstatistically significant in day18compared with day16in ETU malformation group(P<0.05), but obvious difference day20(P<0.01).③Distribution and expression of GDNF, RET protein: The protein of GDNF and RETwere mainly expressed in the cell membrane and cytoplasm of rectum terminal myentericnerve plexus cells and submucous plexus in fetal rats. The expression of GDNF and RETshowed no different among saline control group, ETU control group and ETUmalformation group in day16(P>0.05). The expression of GDNF and RET showed nodifferent between saline control group and ETU control group in day18and20(P>0.05).the expression of GDNF and RET showed statistical significance in saline control group,ETU control group compared with ETU malformation group in day18(41.625±3.888vs23.281±3.993, P<0.01;41.349±1.799vs23.281±3.993, P<0.01;42.000±12.275vs27.643±6.540, P<0.05;41.965±10.374vs27.643±6.540, P<0.05). The expression of GDNFand RET showed statistical significance in saline control group, ETU control groupcompared with ETU malformation group in day20, the difference of GDNF and RETprotein were obviously (165.209±12.35vs48.738±8.262, P<0.01;162.153±13.020vs48.738±8.262, P<0.01;122.107±17.250vs40.807±9.933, P<0.01;125.529±18.754vs40.807±9.933, P<0.01). In saline control group and ETU control group, it showedstatistical significance that the protein of GDNF and RET compared in day16and18(P<0.05), but obvious difference in day20(P<0.01). In ETU malformation group, itshowed no statistical significance that the expression of GDNF and RET in day18compared with day16and20（P>0.05）, but obvious difference between day16and day20(P<0.01).④E xpression of the GDNF and RET: The expression of GDNF and RET showed no different among saline control group, ETU control group and ETU malformation group inthe day16, the difference was no statistically significant (P>0.05). The expression ofGDNF and RET were no statistical significance between saline control group and ETUcontrol group in day18and20. The expression of GDNF and RET showed statisticalsignificance in saline control group, ETU control group compared with ETU malformationgroup in day18(0.706±0.076vs0.478±0.027, P<0.01;0.686±0.068vs0.478±0.027,P<0.01;0.292±0.063vs0.105±0.047, P<0.05;0.293±0.093vs0.105±0.047, P<0.05). Theexpression of GDNF and RET were difference among saline control group, ETU controlgroup compared with ETU malformation group in day20, the difference of GDNF andRET protein were obviously (1.655±0.0625vs0.501±0.030, P<0.01;1.589±0.072vs0.501±0.030, P<0.01;1.333±0.134vs0.208±0.148, P<0.01;1.395±0.194vs0.208±0.148,P<0.01). In saline control group and ETU control group, it was statistical significance thatthe protein of GDNF and RET in day18compared with day16(P<0.05), but obviousdifference with in day20(P<0.01). It showed no statistical significance in3days in ETUmalformation group (P>0.05).⑤E xpression of GDNF mRNA: The mRNA was no different among saline controlgroup, ETU control group and ETU malformation group in day16,the difference was nostatistical significance (P>0.05). The mRNA was no statistical significance between salinecontrol group and ETU control group in day18and20(P>0.05). The mRNA wasstatistical significance in saline control group, ETU control group compared with ETUmalformation group in day18(103.624±27.533vs79.169±11.697, P<0.05;105.184±19.634vs79.169±11.697, P<0.05); The mRNA was difference among salinecontrol group, ETU control group compared with ETU malformation group in day20, thedifference of GDNF and RET protein were obviously (151.496±33.622vs94.873±11.309,P<0.01;150.738±21.423vs94.873±11.309, P<0.01). mRNA was statistical significance inday18compared with day16in saline control group and ETU control group (P<0.05), butobvious difference with in day20(P<0.01). In ETU malformation group, the differencewas obvious between day16and day20(P<0.01).Conclusions:①Expression of GDNF and its receptor RET increase gradually with the constantdevelopment of the embryo in the myenteric and submucosal of distal rectum in normalfetal rats, while decrease gradually in ARM fetal rats, and difference is more obvious withthe constant development of distal rectum; ②Signaling pathways of GDNF/GFRa1/RET may play an important role in adjustingthe enteric nervous system of ARM’s fetal rats; reduce the expression of GDNF and itsreceptor RET may affect postoperative bowel dysfunction in children with ARM;③The ETU is no effect for no teratogenic rat in the development of the entericnervous system;④Days between18-20are vital for the development of enteric nervous system infetal rats.