Neural Lesion In Fetal Rats With Retinoic Acid-induced Myelomeningocele And Fetal Surgery Experiment

BackgroundMyelomeningocele (MMC) is the main cause of neurogenic bladder dysfunction (NBD) in children, which can lead to lifelong physical disabilities, including paraplegia, urinary and fecal incontinence, hydrocephaly, sexual dysfunction, skeletal deformations and mental impairment. Early intervention by fetal surgery could be an effective approach to deal with MMC, although there are still some arguments in the outcome of the surgery. The relationship between spinal cord lesion of fetal MMC and the nervous and muscular modification of the bladder is so far unclear. Therefore, further study is needed by the means of animal models. While a number of surgically created or mutant animal models of MMC exist, they are unable to replicate the primary defect in neurulation, limiting their experimental relevance to the "secondary" injuries of mechanical or chemical trauma. Retinoic acid-induced MMC may be an ideal model.ObjectiveWe want to create and optimize the model of retinoic acid-induced myelomeningocele in fetal rats with higher rate of MMC, lower rate of still birth/absorptions and smaller range of lesion. Based on this model, we want to analyze the development and innervation to the bladder smooth muscle and neural lesion in the spinal cord of the MMC fetus at disparate age, and to reveal the interaction between the lesion of spinal cord and bladder. And several cytokines such as HGF were assessed by RT-PCR in spinal cord of MMC fetal rat and control at different gestational days. Fetal repair of MMC was also tested in this model to explore the possible fetal surgery methods and to see the beneficial effect on the bladder innervations.Materials and Methods1. Optimization of retinoic acid-induced myelomeningocele in fetal rats:Time-dated pregnant SD rats were divided into there groups by the different time of being gavage fed with ATRA dissolved in 2ml of olive oil:group E9, E9.5 and E10, fed at 9 a.m.,11p.m. on the 9th day after pregnancy,9 a.m. on the 10th day after pregnancy respectively. Control rats were fed with 2ml of olive oil alone. Dosage in group E9, E9.5 and E10 ranged in 60-110mg/kg,40-120mg/kg and 80-100mg/kg respectively. Fetus were harvested at E16, E18 or E20 by cesarean section. Numbers of pregnant rats, fetal rats, rats with MMC and still birth/absorptions were accounted. Rates of MMC and still birth/absorptions in treatment group were calculated. Data were analyzed by Pearsonχ2 using Stata 7. Significance was defined as P≤0.05.2. Morphology of nervous lesion in the spinal cord and bladder of fetal rats with myelomeningocele at disparate age:Fetus were divided into MMC group and control group. Each group was further divided into trere groups at disparate fetal age:E16, E18 and E20. A-actin and neurotubulin-β-Ⅲwere analyzed in bladders, GFAP and VAChT were analyzed in lumbosacral levels spinal cords by immunohistochemisty. Photos were taken to determine the integrated optical density of each sample. Data were analyzed by independent t-test using SPSS 16.0. Significance was defined as P≤0.05.3. RT-PCR assess of cytokines such as HGF in spinal cord of MMC fetus and the control at disparate age:Fetus were divided into MMC group and control group. Each group was further divided into 3 subgroup according to gestational days, which is gestational day 18(E18), gestational day20(E20), and gestational day21(E21). In total there are 6 group. In each group, there are 5 fetus. Dissecting microscope were used to dissect whole spinal cord out. RNA was extracted. HGF,it's receptor c-MET, tPA, uPA and GAPDH were underwent reverse transcription and PCR amplification. The data were analysed by SPSS16.0. ANOVA and t test were used for statistical analysis.4. Application of tissue engineering material for fetal repair of MMC:MMC fetus of 18 gestational days were anethesiezed.Midline laparotomy were performed. A small hysterotomy was used to exposed the MMC. Chitosan-gelatin membrane was pasted to the defect by cyanoacrylate adhesive. Fetus was extracted on day 21. HE staining was performed on spinal cord and VACHT and tubulin immunohistochemistry was performed on bladder Photos were taken to determine the integrated optical density of each sample. Data were analyzed by independent t-test using SPSS 16.0. Significance was defined as P≤0.05.Results1. Optimization of retinoic acid-induced myelomeningocele in fetal rats:The rate of MMC was extremely low in group E9 at any dosage, ranging from 0 to 1.92%, while the rate of still birth/absorptions was dramaticly high, ranging from 90.91% to 100%. In group E10, the rate of MMC at the dosage of 120mg/kg was 29.95%, which was the highest in this group. In group E9.5, the rate of MMC at the dosage of 80mg/kg and 100mg/kg were 31.66% and 29.41% respectively, and no significant difference was found. While the rate of still birth/absorptions at 80mg/kg was significantly lower than that at 100mg/kg.At the dosage of 80mg/kg, some fetus with fairly small MMC were gained.2. Morphology of nervous lesion in the spinal cord and bladder of fetal rats with myelomeningocele at disparate age:Neurotubulin-β-Ⅲwas detected at E16 in both MMC group and control group. Whether at E16, E18 or E20, neurotubulin-β-Ⅲin MMC group was significantly lower than that in control group. Numerous of a-actin was detected in both groups at all fetal age. No significant difference was found between MMC group and control group at any fetal age. At E16 and E18, no apparente GFAP positive astrocyte in MMC group or control group was deteced. At E20, in the dorsal region of the spinal cord, numerous GFAP positive astrocytes were detected, with significant difference to the control group VAChT in MMC group was less detected than that in control group at all fetal age with significant difference. At E20, a number of VAChT positive neurons were found in the dorsolateral nucleus in control group.3. RT-PCR assess of cytokines such as HGF in spinal cord of MMC fetus and the control at disparate age:For c-MET, there exist a tendency of stronger expression over gestational days in MMC groups compared with the controlled groups; the difference of MMC group compared with control at gestational day 21 is higher than than the difference at day 18, it is of statisticl significance. For HGF, there also exist a tendency of stronger expression over gestational days in MMC groups compared with the controlled groups; the difference of MMC group compared with control at gestational day 21 is higher than than the difference at day 18 and at day 20, it is of statisticl significance. For tPA the difference between MMC group and the control are of no significance but the expression is higher in MMC. For uPA, here also exist a tendency of stronger expression over gestational days in MMC groups compared with the controlled groups; the difference of MMC group compared with control at gestational day 21 is higher than than the difference at day 18 and at day 20, it is of statisticl significance.4. Application of tissue engineering material for fetal repair of MMC:85pregnant SD rats underwent repair surgery.10 died of anesthetic accident.31 were of MMC defect.1 fetal repair was carried out in one pregnant rat. The fetal repair was successfully carried out in 20 rats. The other 11 failed mainly due to hysterial contraction.7 fetus survived the operation. In 2 of them, the membrane didn't adhere to the MMC,5 fetus is successfully repaired The success rate is 15.1%(over 31 operations). The other 13 feuts died after the surgery. HE stain showed epidermal ingrowth underneath the membrane on day 21. And immunohistochemistry of tubulin in bladder showed similar expression between the normal control and the repair group. The VACHTexpression of bladder in the repair group is similar to the MMC group.ConclusionsGavage feeding to SD rats with 80mg/kg of ATRA at E9.5 can found a successful model of MMC, with high rate of MMC, low rate of still birth/absorptions, and high mimicry to human MMC. The model can be used in further study of fetal surgery of MMC.Differentiation and development of the bladder smooth muscle of MMC fetal rats is morphologically as good as that in normal fetus. Innervation to the bladder smooth muscle of fetal rats takes place no later than E16. Meanwhile, the degeneration of innervation to the bladder in MMC fetus has already occurred. This change synchronously accompanies with the reduction of cholinergic neuron in sacral parasympathetic nucleus and dorsolateral nucleus of the spinal cord.Astrocytes appeared on E18 in fetal MMC model. Numerous of astrocytes emerge in the dorsal region of spinal cord in MMC fetus atE20.RT-PCR assess of HGF and its receptor c-MET suggest:the expression of HGF and c-MET in MMC group over the expression in the normal control group is higher in late gestational day(E21) than in early gestational day (E18), the difference is of statistical significance. It suggests that the expression of HGF and c-MET has a tendency of increasing over the gestational days in MMC group.RT-PCR assess of tPA and uPA suggest:the expression of uPA in MMC group over the expression in the normal control group is higher in late gestational day(E21) than in early gestational day (E18,E20), the difference is of statistical significance. There is no difference for tPA. It suggests that the expression of uPA has a tendency of increasing over the gestational days in MMC group.Chitosan-gelatin membrane could repair the fetal MMC. At gestational day 21 after the repair on gestational day 18, HE stain could find epidermal ingrowth underneath the membrane. Immunohistochemistry of bladder show the tubulin expression is similar to the normal control group, which could support the two-hit theory and suggest the improved bladder innervation.