Comparative Study Of Pelvic Muscles Development In Normal And Abnormal Rats

IntroductionAnorectal malformations (ARMs) are one of the most common digestive disorders, affecting 1/5000 to 1/1500 live births, one kind of malformations supervised by World Health Organization. In spite of the recent advances in operative techniques, the long-term prognosis in intermediate and high type ARMs remains poor. Some patients with ARMs continued to suffer from postoperative anal dysfunctions. The anorectal dysfunction was caused by the dysplasia of the neuromuscular in the pelvic floor. Undoubtedly, the pelvic muscles as the sole muscular sphincter are the focal point of the organs of continence. It is considered that the pelvic muscles are one of the most important factors that influence defecation function.In recent studies, it is suggested that in intermediate and high type ARMs, there are some differences in the skeletal muscle around the anorectum and smooth muscle of anal canal. However, until now, the embryonic development and pathological changes of ARMs continue to be poorly understood and controversial.With the development of molecular biology, the embryogenesis of muscle has been illuminated initially. Myogenic Regulatory Factors (MRFs) play a crucial role in the development of muscle. The muscular precursor cells have followed the pathway regulated by MRFs to differentiate. Wnt signaling pathway regulates MRFs by the classical signal pathway and /or kinase-dependent signal pathway during the embryogenesis. It is implied that Wnt signal pathway may affect the MRFs by changing the conformation of chromatin. The function of Wnt signal pathway for regulating the development of muscle is network modulation rather than linear. In this paper, the ETU murine model of ARMs was employed. The embryogenesis of the striated muscle complex (SMC), the external anal sphincter(EAS) was continuously studied by H&E staining and immunolabeling to Myogenin/MyoD. The immunohistochemistry and RT-PCR were used to detected the expression of Wnt1,MyoD,Myogenin during the development of pelvic muscles in the normal and abnormal rat embryos.α-Smooth Muscle Actin(a-SMA), a specific antibody to smooth muscle, was involved to explore the development of smooth muscle of the termination of digestive tract in the normal and abnormal rat embryos. The embryogenesis of pelvic muscles that influence defecation function and the related factors that influence the development of pelvic muscles were initially illuminated.Material and MethodsAnimals: Wistar rats (230-250g) were provided by Medical Animal Center in Shengjing Hospital of China Medical University.Reagents:①The specific antibodies to Myogenin,α-SMA were purchased from Neomarker Corporation.②The specific antibody to Wnt1 were purchased from Santa Cruz Corporation.③The S-P kits for immuhistochmistry were purchased from MaiXin biological limited company, FuJian Province.④Reverse Transcription kits were purchased from TAKARA biotechnology (DALIAN) CO., LTD.Time-mated pregnant Wistar rats were gavage-fed a single dose of either 125 mg/kg of 1% ETU (2-Imidazolidinethione, 98%; Aldrich Chemical Co, Inc, Germany) or an equal dose of saline on gestational day 10 (E0—sperm in vaginal smear after overnight mating). The embryos were harvested via cesarean section on E13 to E16 (the period of hindgut development) and E17, E18, E21 (term period). The embryos were fixed in 4% paraformaldehyde/0.1 M phosphate buffered saline (PBS) for 12 to 24 hours depending on their size. Then the embryos from each age group were dehydrated, embedded in paraffin and sectioned serially transversely, sagittally and coronally at 4μm thickness.①The embryogenesis of SMC and pars EAS was continuously studied by H&E staining and immunolabeling to Myogenin.②Wnt1,MyoD,Myogenin immunohistochemistry analyses were carried out to study the expressions in the pelvic muscles by measuring the ratio of the positive area.③α-SMA immunohistochemistry was employed to explore the embryogenesis of internal anal sphincter (IAS) and to make a comparison between the normal and abnormal embryos. Moreover, the hindgut and pelvic muscles were frozen in liquid nitrogen then to -80℃and dissected under magnification. The specimens were used for total RNA extraction. Wnt1, MyoD, Myogenin andα-SMA expression was evaluated by RT-PCR. One-way ANOVA was used to compare the differences of foresaid genes expression levels between the normal and abnormal groups.ResultsFrom E16 on, it was observed the fibroid structure in the pelvic floor of the rat embryos. On E16, SMC loosely surrounded the anal canal of the normal embryos. In ARMs rats, the location and appearance of SMC were similar to the normal group. From E18 on, SMC, which thickened obviously, surrounded the rectal terminus more tightly than on E16 in the normal group. However, the obvious changes of SMC, which was characterized by abnormal location, appearance and path, could be noted in ARMs rats. In detail, SMC shifted obviously cephalad, ventrally and medianward, and converged inferior to the rectal terminus and posterior to the urethra. Under high-power view, there was connective tissue among inter-muscular bundles, and the structure of SMC was disordered. During the following gestational days, SMC in the normal and ARMs groups continued their own tendency, respectively.The myogenic precursor cells of the external anal sphincter could be noted on the perineal position subcutaneous part from E17 on in normal group, however, the foresaid musculature could be observed on the subcutaneous part which was superior to the proctodeum in the abnormal group. Although the development of the EAS was lagged behind, the musculature of that was thin, it grew fast after birth. The lateral musculature shifted cephalad. On the postnatal 2 weeks, the lateral musculature of those muscles reached the lateral of the IAS on the saggital sections. At the same time, the rectal longitudinal muscle insert into the muscle bundle of the IAS and the EAS. The three dilatate musculatures interlaced with each other and composed the muscles complex.The immunohistochemistry results of Wnt1: Wnt1 positive cells could be observed in the original area of pelvic muscles from E13 to E21. Before E16 (including E16), the positive myogenic precursor cells in the normal group were more significant than the abnormal group; from E16 on, there was no significant difference between the two groups. Wnt1 mRNA RT-PCR results: Wnt1 gene expression levels in the normal group was higher than that in the abnormal embryos from E13 to E15, moreover, there were significant differences between the two groups. However, on E16,E18 and E21, there was no difference between the two groups.The immunohistochemistry results of MyoD: The positive cells could be observed in the original area of pelvic muscles from E13 to E16. MyoD expression was negative in the pelvic muscles after E16. The positive myogenic precursor cells in the normal group were more significant than the abnormal group. MyoD mRNA RT-PCR results: The expression levels of MyoD in the ARMs embryos was lower on E13-E16 than that in control group (P<0.05). The immunohistochemical results were consistent with the RT-PCR results.The immunohistochemistry results of Myogenin: From E15 to the term period, there were positive expressions in the pelvic muscles and SMC, and there were no statistic differences between the two groups. The expression of Myogenin in subcutaneous part of the EAS commenced on E17, and the tendency continued until the term period; furthermore, there was no statistic difference between the two groups. Myogenin mRNA RT-PCR results shew that Myogenin expression levels increased on E15-18, but decreased on E21. There are no significant differences between the two groups on every age. The immunohistochemical results were consistent with the RT-PCR results.From E15 on, theα-SMA immunolabeling Myogenic precursors of Myogenic precursors of smooth muscles could be noted on the primitive anorectum, and then the precursors of circular muscle was generated by them. In the normal group, the circular muscles of the primitive anorectum thicken slightly, and shaped a little dilatate musculature.The circular muscle bundles, which is composed the primordium of the IAS, arranged like the overlapping and continuous watts. The end segment of the muscular fiber slightly sloped upward slightly, the middle segment gradually showing horizontal, and the superior segment sloped intero-inferiorly. In the ARMs group, theα-SMA immunolabeling Myogenic precursors of the smooth muscles could be noted on the primitive anorectum, as well. The musculature was similar to the normal group.α-SMA expressed in the following embryogenesis. On E15 and E16, the expression ofα-SMA reached the peak level, and there was no significant difference between the two groups. On E17, E18 and E21,α-SMA expression in the normal embryos was a little higher than that in the ARMs embryos. On E16,α-SMA immunolabeling longitudinal muscles of the rectum could be observed on the lateral circular muscles. With the development, the muscles was asynechia.At the term, the whole longitudinal muscle was shaped successively but very thin. In the normal group, at the end of gestation, the longitudinal muscle gave off some fibers inserted into the IAS and the EAS. In the abnormal embryos, although the longitudinal muscle gave off some fibers inserted into the circular muscle, there was some distance between the EAS. After delivery, the IAS and longitudinal muscle developed more rapidly. At the postnatal 2 week, there was an evident dilatate musculature on the termination of the IAS and longitudinal muscles. The longitudinal muscles gave off fibers insert into the IAS and the EAS,and formed the muscles complex, which was composed of the longitudinal muscles, the IAS and the EAS. The RT-PCR results were consistent with the immunohistochemical results.ConclusionsThis study illustrated the development of the SMC in the normal and ARMs rats. On E16, the location and appearance of SMC in ARMs rats were similar to the normal rats. And at this time, the ectopic rectal orifice could be noted. From E18 on, the maldevelopment of SMC could be observed in ARMs rats. These observations suggested that the morphological changes of SMC took place after the occurrence of abnormal anorectum.The data, thus, indicated that Wnt1 and MRFs molecules play an important role in anorectal pelvic muscles morphogenesis and the decreased expression of Wnt1 and MRFs might be related to the dysplasia of the neuromuscular in the pelvic floorα-SMA immunolabeling primitive anorectal smooth muscles could be observed on E15. The longitudinal muscle development was later than the circular muscle. There was IAS, which surrounded the termination of the ectopic anal canal in the ARMs embryos. The development of the IAS in the ARMs group was a little dysplasia than that in the normal group. The internal anal sphincter and the longitudinal muscle developed notablely within the postnatal 2 weeks.