Apoptosis during the development of pelvic floor muscle in anorectal malformation rats☆

Abstract 

Purpose

Fecal incontinence and constipation still remain as major postoperative complications after procedures for anorectal malformations (ARM). The striated muscle complex (SMC) is one of the most important factors that influence defecation. Previous studies have demonstrated different degrees of the muscle complex dysplasia dependent on the complexity of ARM. To explore the mechanisms of maldevelopment of SMC in ARM, apoptosis was investigated during pelvic floor muscle development in rat embryos with ARM.

Methods

Anorectal malformations in rat embryos were induced by treating pregnant rats with ethylenethiourea on the 10th embryonic day (E10). Normal and ARM rat embryos from E16 to E21 were serial-sectioned transversely or sagittally, and SMCs were dissected and snap frozen. TdT mediated dUTP Nick Ending Labeling (TUNEL) staining and DNA ladder analysis were performed to identify apoptosis and expression of Bax/Bcl-2 were confirmed with immunohistochemical staining and Reverse Transcription-Polymerase Chain Reaction (RT-PCR) analysis.

Results

Hypoplastic and disordered SMC sling shifted cephalad, ventrally, and converged inferior to the rectourethral fistula and infiltrated connective tissue in ARM embryos. In the normal group, TUNEL-positive cells became evident on E17; sporadic positive staining was mainly localized in 2 areas as follows: the junction area between SMC and bulbocarvernosus muscle and posterior to the rectum where bilateral SMC converged. In the ARM group, massive positive staining of nuclei was observed from E16 to E21 and was mainly distributed in the dorsal part of the SMC. Electrophoresis of DNA samples yielded a “ladder” pattern of migration both in normal and the ARM group from E17 to E21, the ladders were stronger in the ARM group. In both groups, the expression of Bax/Bcl-2 was detectable on E17, the immunoreactivity increased on E19 and E21. Compared with the normal group, the expression of Bax was increased, whereas Bcl-2 was declined in the ARM group. Significant upregulation of Bax messenger RNA (mRNA) levels and downregulation of Bcl-2 mRNA levels were observed in ARM embryos.

Conclusions

In the current study, abnormal apoptosis and disturbed expression of Bax/Bcl-2 were identified during SMC development in ARM embryos. It is suggested that precocious, excessive, and dislocated apoptosis might be a fundamental pathogenesis for the maldeveloped SMC in ARM rats. The temporospatial expressions of Bax/Bcl-2 indicate they may have an important role in the regulation of apoptosis of SMC.

Key words: Anorectal malformations, Striated muscle complex, Apoptosis, Bax/ Bcl-2, Embryogenesis