The Study Of Cloacal Apoptosis And Intestinal Interstitial Cells Of Cajal In Anorectal Malformation

The study of Cloacal Apoptosis and Intestinal Interstitial Cells of Cajal in Anorectal MalformationObjectiveAnorectal malformation(ARM) are very common pediatric surgical disorders affecting 1 in 5,000 to 1 in 1,500 live births. The spectrum of these abnormalities is very complicate and the complications are very much which affect pediatric health.Despite a long history of normal embryological research, the developmental processes of anorectum remain contentious. In embryological developments, apoptosis are deeply involved in the descending of urogenital septum(URS), the configuration transformation of cloaca and the rupture of cloacal membrane. Presently, the role of apoptosis in cloaca remains unclear in the embryological developments of ARM. In this study, Wistar rats were treated by ethylenethioureal(ETU) and gave birth to embryos with ARM. We studied the spatiotemporal distribution of cloacal apoptotic cells as well as the cloacal expression of apoptotic genes bcl-2 and bax in abnormal embryos of gestation day 13 to 16.In 1980s, posterior sagital anorectoplasty provided an optimal access to reconstruct musclecomplex in ARM. The rate of faecal incontinence descended significantly, while the rate of constipation increased apparently. Some patients with severe constipation needed one or more operations, which affected their quality of life. Recently, interstitial cells of Cajal(ICC) were regarded as electrical pacemaker and mediated enteric neurotransmission. Abnormal development of ICC lain in some gastrointestinal functional diseases. The development of ICC in ARM are unclear. We studied the entire intestinal development of ICC in rat embryos with ARM and the expression of genes c-kit and SCF in the terminal rectum of patients with ARM.Methods1,To detect the spatiotemporal distribution of apoptosis in cloaca of normal and abnormal rat embryos from gestation day 13 to 16 with TUNEL staining.2,To detect the spatiotemporal expression of gene bcl-2 and gene bax in cloaca of normal and abnormal rat embryos from gestation day 13 to 16 with SABC staining.3,To detect the expression of ICC in intestine of normal and abnormal rat embryos on gestation day 21 with SABC staining.4,To detect the expression of gene c-kit and gene SCF in terminal rectum of patients with ARM with RT-PCR.Results1,Spatiotemporal distribution of cloacal apoptotic cells in ARM On embryonic day(E) 13, the genital tubercle had developed and the cloaca appeared clearly. The URS divided cloaca into urogenital sinus and rectum. The tail groove had formed. There were apoptotic cells in the epithelial layers and mesenchyme region of URS. Following the development, genital tubercle growed gradually and shifted ventrocaudally. The tail groove deepened gradually and cloacal membrane rotated dorsalcaudally. Apoptotic cells increased and descended in the mesenchyme region of URS. Many apoptotic cells appeared in the dorsal mesenchyme of the rectum. On E14, apoptotic cells began to appear at the terminal rectum and the future anal orifice part of cloacal membrane. On E15, URS fused with cloacal membrane and apoptosis in URS descended to the fusion position. Anal membrane ruptured and rectum communicated with the outside on E16.In embryos with abnormal cloaca, the development of genital tubercle, the descent of URS and the configuration transformation of cloaca dalayed. Apoptotic cells decreased apparently in URS, the dorsal part of rectum and the cloacal membrane. On E15, the fusion of URS and cloacal membrane did not appear. On E16, anal membrane did not rupture which resulted in ARM.2,Spatiotemporal expression of gene bcl-2 and gene bax in the cloaca of ARMOn E13, over 80% of epithelial cells in URS were bax positive cells. Less than 10% of mesenchyme cells in URS were bax positive cells. Follwing the development, positive cells in epithelial cells decreased. But on E14, positive cells in mesenchyme increased significantly and positive cells appeared at the future anal orifice part of cloacal menbrane. On E15 and E16, positive cells of mesenchyme region of URS were few. Comparing with normal embryos, bax positive cells were fewer in the epithelial layers and mesenchyme region of URS in abnormal cloacal embryos on E13.5 and E14.On E13, bcl-2 positive cells were more apparent in the epithelial layers of URS. Then they decreased gradually. Positive cells in mesenchyme region of URS were similar from E13 to E16, less than 10%. But in abnormal cloaca, bcl-2 positive cells were abundant in the epithelial layers and mesenchyme region of URS on E13.5.3,The expression of ICC in the entire intestine of ARMc-kit positive cells distributed mostly around the myenteric plexus region of the small intestine, colon and rectum. There were a few positive cells in the muscularis.In normal embryos of E21,there were abundant c-kit positive cells around over 80% of the myeteric plexus in the small intestine, colon and rectum. In embryos with ARM, positive cells in small intestine were similar, but positive cells were fewer in colon and rectum compared with normal embryos.4,The expression of c-kit and SCF in terminal rectum of patients with ARMThe expression level of c-kit mRNA in control group,high ARM,intermedia ARM,low ARM and acquired anal fistula were 1.79±0.22,0.67±0.19,0.89±0.17,1.57±0.25,1.68±0.18, respectively. There were significant difference of expression level in high and interrnadia ARM groups (P＜0.05) and no significant difference in low ARM and acquired anal fistula groups (P＞0.05) compared with control group. The expression level of SCF mRNA in control group,high ARM,intermedia ARM,low ARM and acquired anal fistula were 1.72±0.19,0.65±0.16,0.98±0.19,1.61±0.24,1.69±0.14, respectively. There were significant difference of expression level in high and intermadia ARM groups (P＜0.05 ) and no significant difference in low ARM and acquired anal fistula groups (P＞0.05) compared with control group.Conclusions1,In anorectal embryological developments, apoptosis were deeply involved in descend and fusion of URS, the configuration transformation of cloaca and the rupture of cloacal membrane. Apoptotic cells decreased apparently in URS, the dorsal part of rectum and the cloacal membrane with ARM embryos. These hampered the normal anorectal embryological development and might result in ARM.2,The gene bcl-2 and gene bax were involved in cloacal apoptosis. The abnormal expression of gene bcl-2 and gene bax in the epithelial layers and mesenchyme region of URS in malformation embryos might result in abnormal apoptosis.3,On E21, abnormal development of ICC in the colon and rectum with ARM embryos might affect the building of neonatal ICC net. This might be one of the reasons resulting in post-operative constipation.4,The expression level of c-kit mRNA and SCF mRNA decreased in high and intermadia ARM groups. These might be congenital and damage the development and functions of ICC. This phenomenon might be one of the biomolecular evidences which resulted in post-operative constipation.