Relationship Between Nitric Oxide Synthase And Neural Tube Defects

ObjectiveNeural tube defects(NTD) are a common congenital malformation in humans, which have affected on the human quality and are the most severe desease of birth defects, which comprises a series of defects such as anencephalia, encephalocoele, myeloschisis and so on because of no and delay closure of the neuropore in the progress of developing embryo.NTD are the multi-factors in the etiology, are caused by both genetic factors and environmental factors. Neural tube was formed by the apophysis,bend and close of neural crest. The top of neural tube forms basic encenphalic shape,namely endbrain, interbrain, midbrain and afterbrain. The abnormal closure of neural tube will result in neural tube detects. Encephalocoele is a common congenital malformation whose mechanism was no known. NO participates in the physiologic process of brain. In the process of development,NO can promote the differentiation of nervous system by cell apoptosis and promote both the growth and contact of nerve cell. In ECs,NO is a product of the conversion of L-arginine by endothelial NO synthase,which determines the biologic domino offect of NO. NOS isoforms-neuronal (nNOS), endothelial (eNOS),and induciable(iNOS) were found in the brain.NO induce apoptosis in the early NT development. However,it is yet unclear to which types of NOS play a dominant role in NT development. The present study was undertaken in an attempt to determine if morphogenesis malformation of the NTD could be induced in wister rat by administration of ETU.The morphogenesis of encephalocoele in rats given ETU was investigated by light microscopy. From mutually affect on endothelial cell and the neuron, We explored the molecule bases of changes in encephalocoele through studied expression of eNOS, nNOS, iNOS protein and mRNA. And illuminated the effect of eNOS, nNOS, iNOS on brain in neural tube defects. Materials and methodsMature Wister rats, aged frome 2 to 3 months and weighing approximately 300g,were used in this experiment. sixty female(Animal Laboratory.china medical university)were time mated with a male rat overnight.The day on which sperm was found on vaginal smear was designated as day 0 of gestation.The pregnant rats were divided randomly into control (10rats) and experimental group (50 rats).After pregnant, experimental rats received 1% ETU(125mg/kg) by garage on gestation day 10.Control rats received only the vehicle at the same volume. All dams received cesarean section on gestation day 14,20.Fetuses aged E20 brains were perfused via the left ventricle,first with heparin (15,000IU/I)in 0.1 M potassium phosphate-buffered saline(KPBS)for half a minute,and then with 4% paraformaldehyde and immersed in 30% sucrose-KPBS overnight at 4℃,and processed for paraffin sectioning or cut into 10-um cryostat sections.Some brain were stored fluid nitrogen and then stored at-70℃refrigerator.Sections were stained with H￡E for light microscopic examination.1. H￡E staining was carried out in cerebral cortex of rat embryo to determine the pathological changes of rat cerebral cortex in animal model with neural tube defects(NTD).2. In situ Cell Apoptosis Detection (Tunel)Sections are examined by Tunel in order to observe the apoptosis cells in E20 cerebral cortex of all groups.3. The immune-fluorescence method was used to test the distribution and expression of bcl-2 in cerebral cortex of rat embryos; Western blot analysis was performed to determine the semiquantitative of caspase-3 and bcl-2 protern expression.4. The immune-fluorescence method was used to test the distribution and expression of NOS in cerebral cortex of rat embryos;Western blot analysis was performed to determine the semiquantitative of nNOS,eNOS and iNOS protern expression.5. Brain RNA was extracted in cerebral cortex of rat embryo by guanidine isothiocyanate-phenol-chloroform method.Tagman quantitative real time PCR method was used to analysis the quantitative of NOS mRNA in animal model with neural tube defects. Results1. NTD animal model:NTD can be produced by intragastric administration of ETU to timed-pregnant rats:about 80% offspring have NTD. The volume and cells of cerebral cortex were thinner in encephalocoele embryos than in other ETU-injected and in control rat embryos.And there were no evidence changes in histopathologic analysis of brain tissues in other ETU-injected and in control rat embryos.2. Results of In situ cell apoptosis detectionIn E20 cerebral cortex, more Tunel(+) cells were found in encephalocoele group than control group,and the results were significant.3. Expression of Bcl-2 in cerebral neronsIn encephalocoele groups,the bcl-2 immunoreative signal showed trace staining in E20 cerebral cortex compared with other groups.4. Expression of Caspase-3 and bcl-2 proteinIn E20 cerebral cortex, the expression of cleaved Caspase-3 in the encephalocoele group are higher than that in control group, and the expression of bcl-2 in the encephalocoele group are lower than that in control group. These findings were consistent with the results of the western blot analysis in E14 Brain.Comparison of gray values both Caspase-3,bcl-2 and GAPDH was done through Band Scan analysis soft, and the results were significant.5. Expression of nNOS,eNOS and iNOS protein:In encephalocoele groups,the nNOS immunoreative signal showed trace staining in E20 cerebral cortex compared with other groups. The eNOS immunoreative signal showed no change in E20 cerebral cortex in all groups,But the more numbers of eNOS (+) vessel were found in encephalocoele groups than that in other groups. iNOS wasnot detected in all groups. These findings were consistent with the results of the western blot analysis.Western blot analysis confirmed that lower expression levels of nNOS and higher expression of eNOS in E14 brain tissue in the encephalocoele group compared with the control group. These findings were consistent with the results of E20 cerebral cortex. iNOS was detected in E14 brain and There were no evidence changes of expression of iNOS in the encephalocoele group compared with the control group.6. Expression of nNOS mRNA, eNOS mRNA and iNOS mRNA in the E 20 cerebral cortex:There were no obvious changes of expression of nNOS mRNA in ETU-injected and control groups. Expression of eNOS mRNA increased significantly in cerebral cortex of the encephalocoele group, And there were no obvious changes of expression of eNOS mRNA in cerebral cortex of the control group and the other ETU-injection groups. iNOS mRNA weren't dectected in all groups.Conclusion1. Pregnancy rats treatment with ETU on gestation 10 successily induced NTD.2. The neurons apoptosis induced by eNOS-secreted NO may be involved in caspase-3, bcl-2 singaling pathway in the encephalocoele group, which leads to neural tube closure defects. It suggested that eNOS played a dominant role in the process of NTD.3. The unbalanced proportion between vessels and neurons could resposible for NTD.