| Objective:Anorectal malformations（ARMs）are the most frequent congenital anomalies,with incidence ranges approximately 1 per 1500 to 5,000 live births in pediatric surgery,accounting for 25%of gastrointestinal malformations.Despite recent improvements in the treatment and postoperative care of ARMs,post-operation complications are increasingly recognized as a serious,worldwide public health concern.There are at least 1/3 patients with ARMs still suffered from some chronic symptoms after operations,particularly with fecal incontinence and constipation,which severely influence the mental and physical health of the children and bring patients,their family and the society serious burden.The causes of poor postoperative anorectal function are still obscure and have been attributed to many factors,and researchers have realized that lumbosacral spinal cord malformations are a major leading cause of post-operation complications.Our previous study has demonstrated that abnormal development existed in lumbosacral spinal cord of ARMs,which is a main reason for post-operation defecation obstacles and put forward a new therapy about recovering the central nervous function of defecation.Research has confirmed ARMs patients and rats model are always complicated with different extent of lumbosacral spinal cord dysplasia.Although the importance of lumbosacral spinal cord anomalies in ARMs has been recognized,research is still in the initial stages.Previous studies about ARMs patients demonstrated that the motoneurons in the median ventral horn of high or intermediate deformity are fewer than that of the normal and sacral myelodysplasia is one of the neuropathological features.Previous studies about ARMs model demonstrated that neurons in the lumbosacral spinal cord innervating the levator ani and anorectum were decreased in number and dysplasia,which was one important feature of neuropathological deficiencies.So far,however,it is not clear that the embryogenesis mechanism of the ARMs is abnormal.Therefore,in this study,high-throughput sequencing was used to screen the genes of abnormal expression in the lumbosacral spinal cord of ARMs in the whole transcriptome,and to investigate the mechanism of abnormal development of neurons.The development of spinal cord requires the participation of a series of cell activities such as proliferation,migration,differentiation and apoptosis,and these cell activities are strictly regulated by a complex network of multiple signals.The roof plate in the dorsal midline,and the floor plate and notochord in the ventral midline,establish dorsoventral identities;with Bmp and Wnt signaling being instrumental in inducing the roof plate and specifying dorsal patterning,and Shh signaling being instrumental in inducing the floor plate and specifying dorsal patterning.BMP and WNT was expressed in a high to low concentration along the dorsal-ventral axis,causing nerve cells to differentiate into the middle neuron.While Shh was expressed in a low to high concentration along the dorsal-ventral axis,essential for the generation of distinct progenitor cells,and it promotes the production of motor neurons and oligodendrocytes from ventral cord progenitor cells in the lumbosacral spinal cord.Members of BMP、WNT and Shh are synergistic or antagonistic to maintain the migration,differentiation,proliferation,axon orientation,and target organs of various nerve cells.Changes in any part of the process can lead to abnormal development of neurons.During spinal cord development,apoptosis is critical for eliminating excess neural progenitor cells in the nerviduct and neurons that fail to make effective synaptic connections.This process facilitates quantitative matching of neurons with their targets,which is essential for normal nervous system morphogenesis and function.According to“Neurotrophic Factor hypothesis”,the number of neural cells is controlled by the availability of their innervating targets,we speculate that excessive apoptosis in lumbosacral spinal cords was due to dysplasia of target organ which can’t exert enough neurotrophins to combine with the neural cells in CNS.This process eliminated more“useless”neural cells and too few neural cells survived.This result was consistent with previous studies which have confirmed abnormal apoptosis of PFMs and maldevelopment of striated muscle complex and internal anal sphincter during embryogenesis of ARMs rats.Besides,our previous studies have shown that neuron in lumbosacral spinal cord governing striated muscle complex and internal anal sphincter deceased significantly.At present,whether the deceased number of neuron in in lumbosacral spinal cord are due to apoptosis is not clear,we speculate that excessive apoptosis play an important role in the maldevelopment of lumbosacral spinal cord in ARMs rats.Finally,with excessive neural cell apoptosis,fewer synaptic connections might be made,leading to poor biosignal processing and reflex latencies.These may be key factors for abnormal development of ARMs rats.This research used MSCs transplation into lumbosacral spinal cord of ARMs rats to explore a new therapy method for reparing lumbosacral spinal cord dysplasia.The repair of nerve damage is extremely difficult,in recent years stem cell research has made great progress in spinal cocrd injury and degenerative disease of nervous system,however,there were seldom studies on congenital diseases.Many studies have shown that the interaction between MSCs and nerve cells in the settlement area leads to the production of some cytokines,such as neurotrophic factors,interleukin,stem cell factors,and so on.These cytokines promote the recovery of nerve function.MSCs can also produce self-secretion and paracrine factors which are important for the development of pedigree.It can also migrate to pathological tissue and location-specific differentiate to nerve cells,which is beneficial to the repair of damaged nerves.The characteristics MSCs can interacte with nerve cells in settled areas,are very suitable for the treatment of spinal cord dysplasia with ARMs and promising treatment strategies to improve postoperative dysfunction of ARMs.In this paper,we applied the Wistar rat animal model of ARMs induced by ethylenethiourea（ETU）to investigate the development process of lumbosacral spinal cord in normal and ARMs rat embryos.We used“Illumina HiSeqTM2000 sequencing technique"experiment to screen differentially expressed transcriptome of lumbosacral spinal cord tissue in normal and ARMs rat embryos.The immunohistochemistry,RT-qPCR and Western Blotting were applied to analyzed the expression of Bcl2/Bax and Shh-Ptch1-Gli1 and TUNEL was applied to analyzed nerve cells apoptosis during the development of lumbosacral spinal cord in the normal and ARMs rat embryos.Besides,MSCs were isolated from Wister rat femoral,and transplanted to detective lumbosacral spinal cord in ARMs rat embryos after in vitro culture and proliferation.Then we observed the differentiation of nerve cells in lumbosacral spinal cord in rat embryos with ARMs after transplantation,to explore the treatment potential of MSCs transplantation in the defective lumbosacral spinal cord of rat embryos with ARMs.Methods:1.Animal model and tissue collection.Ten-twelve weeks of Wistar rats（250-300 g）were obtained from the Experimental Animal Center,Shengjing Hospital of China Medical University.Male and female（ratio 5:l）were mated overnight,the appearance of sperm in vaginal smear at the morning after mating was determined as embryonic day 0（E0）.The pregnant rats were fed by stomach tube a single dose of either 1%ethylenethiourea（ETU;125 mg/kg）to induce ARMs（ARMs group）or an equal dose of saline（normal group）at E10.The embryos were harvested via cesarean section on E16,E17,E19 and E21,and were fixed in 4%paraformaldehyde solution.Then the tissues from each age group were dehydrated,embedded in paraffin,and sectioned sagittally at 4μm thickness.For western blotting and RT-qPCR analyses,lumbosacral spinal cord tissues were removed under a light microscope and stored at-80?C.2.Preliminary screening of the differentially expressed transcriptome（Shanghai bohao biological technology co.,LTD）was used to screen differentially expressed transcriptome of lumbosacral spinal cord tissue and predict their interactions in normal and ARMs rat embryos on E17.Then RT-qPCR analyses were applied to confirm parts of the differentially expressed RNAs on the development of lumbosacral spinal cord.3.Spatiotemporal expression of Shh-Ptch1-Gli1 during the development of the lumbosacral spinal cord.The expressions pattern of Shh-Ptch1-Gli1 mRNA in the normal and ARMs rat embryos were detected by RT-qPCR analysis.Spatiotemporal expressions of Shh-Ptch1-Gli1 proteins were confirmed by immunohistochemistry staining.Western blotting was also used to discover the expression difference of Shh-Ptch1-Gli1 proteins between the normal and ARMs rat embryos.4.Spatiotemporal expression of Bcl2/Bax and nerve cells apoptosis during the development of the lumbosacral spinal cord.TUNEL staining was performed to identify apoptosis and spatiotemporal expressions of Bcl2/Bax proteins were confirmed by immunohistochemistry staining.Western blotting was also used to discover the expression difference of Bcl2/Bax proteins between the normal and ARMs rat embryos.The expressions pattern of Bcl2/Bax mRNA in the normal and ARMs rat embryos were detected by RT-qPCR analysis.5.Ability of in utero MSCs transplantation in repairing the defective lumbosacral spinal cord.MSCs were isolated,cultured and expanded in vitro.Then MSCs were transplanted into the defective lumbosacral spinal cord in rat embryos with ARMs by fetal surgery and microinjection.The differentiation and apoptosis of nerve cells was observed in defective lumbosacral spinal cord of rat embryos with ARMs.6.Statistical analysis.Allnumerical data were presented as mean±standard deviation（SD）.A two-sided t test was used for the comparison of two groups,and a one-way ANOVA and SNK post-hoc test was used for multiple groups.The Pearson correlation was calculated with the use of SPSS 13.0.p<0.05 was consideredstatistically significant.Results:1.The results of“Illumina HiSeqTM2000 sequencing technique”experiment（screening conditions,Fold Change cut-off:2.0,p value cut-off:0.05）showed that compared to normal group,the expression of 53 mRNA,63 lncRNAs and48 circRNAs were decreased and 41 mRNA,49 lncRNAs and 31 circRNAs were increased in ARMs group on E17.The verification results showed that the results trends between the qRT-PCR analysis and expression profile chip were basically identical.2.The results of RNA-Seq for transcriptome suggested the expression of Shh was abnormal,and the further research about the spatiotemporal expression study of Shh-Ptch1-Gli1 in the development of lumbosacral spinal cord between normal group and ARMs group have been performed.We found that in the normal group,from E16to E17,Shh/Ptch1 expression in lumbosacral spinal cord increased significantly and reached the peak on E17,while Gli1 reached the peak on E21.Positive staining cells were ventral part of lumbosacral spinal cord.Similar change trends in Shh-Ptch1-Gli1expression were detected in ARM model rat embryos;however,the expression of Shh-Ptch1-Gli1 was signifcantly reduced compared with the normal rat embryos.3.The results of RNA-Seq for transcriptome suggested the expression of Bax was abnormal,and the further research showed that apoptosis index（AI）in the ARMs group was significantly higher compared to normal group and reach the peak on E19.The spatiotemporal expression study of Bcl2/Bax in the development of lumbosacral spinal cord between normal group and ARMs group has been performed.Our results showed that the expression of Bcl-2 decreased,whereas the level of Bax increased in the ARMs fetuses.In addition,there was a significantly negative correlation between protein expression of Bcl-2/Bax ratio and AI in the ARMs group.4.The extracted MSCs were cultured for 48h,passaged to P3-P6 and then transplanted into lumbosacral spinal cord of ARM model rat embryos.The spatiotemporal expression study of Synapsin and GFAP and apoptosis of neural cells in the development of lumbosacral spinal cord between normal group,ARMs group and MSCs+ARMs group have been performed.Compared with the normal group,the expression of Synapsin decreased in ARMs group whereas after transplantation the expression of Synapsin increased in MSCs+ARMs group,but there was significantly difference between the normal group and the MSCs+ARMs group.Compared with the normal group,the expression of GFAP and apoptosis of neural cells increased in ARMs group whereas after transplantation the expression of GFAP and apoptosis of neural cells decreased in MSCs+ARMs group,but there was significantly difference between the normal group and the MSCs+ARMs group.Apoptosis index（AI）in the ARMs group was significantly higher compared to normal group.AI in MSCs+ARMs group is significantly lower then that in ARMs group but still significatntly higher than that in control group.Conclusions:1.The development of lumbosacral spinal cord during embryonic phase is a very complex process.The results of RNA-Seq for transcriptome suggested that expressions of many mRNA and ncRNAs were very different between normal group and ARMs group,and many signaling pathways were involved.These RNAs and signal transduction pathway might be the cause of abnormal development of lumbosacral spinal cord in rat embryos with ARMs.2.From E16 to E21,the expression of Shh-Ptch1-Gli1 is mainly distributed in the ventral part of the lumbosacral spinal cord.Compared with the normal group,expression of Shh-Ptch1-Gli1 are down-regulated in both mRNA and protein levels in ARMs group,suggesting that lower expression of Shh-Ptch1-Gli1 might lead to the maldevelopment of lumbosacral spinal cord in rat embryos with ARMs.3.From E16 to E21,Apoptosis index（AI）in the ARMs group was significantly higher compared to normal group and TUNEL-positive cells were mainly localized in the ventral horn,the expression of Bcl2 decreased,whereas the level of Bax increased in the ARMs fetuses.The negative correlation between the ratio of Bcl2/Bax and AI manifested that Bcl2/Bax pathway might be the mechanism for neural cell apoptosis in ARMs.4.MSCs,after being implanted into lumbosacral spinal cord,showed the expression of Synapsin increased and GFAP and amounts of neural cellular apoptosis decreased in ARMs group.These results demonstrated strong ability to cause local cell proliferation and differentiation by affecting local microenvironment and signaling molecules.They are suitable to recover lumbosacral spinal cord of ARMs rat fetus,which should be a promising method in improving fecal incontinence following anorectal malformations reconstruction. |
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