Study Of The Mechanism Of Spina Bifida In Fetal Rat

Neural tube defects ( NTDs) are among the most common congenital malformations and supervised by World Health Organization , they occur worldwide with an incidence of between 1 and 9 per 1000 births. Development of central nervous system (CNS) is a complicated progress which is effected by both genetic and environmental factors. The mechanism of NTDs is unknown now, man-y genetic and environmental factors are implicated. NTDs posses the genetic tendency, the risk of recurrence in a sibship is about 2-5%.NTDs consist of a kind of malformations of brain and spinal cord, which are various and usually combined with multiple system anomalies. So it is necessary to induce the NTDs model and to study the etiology and mechanism by the point of view of the embryogenesis and biotomy.The contents of the study are below: (1)animal model; the pregnant rats were divided into 5 groups at random: the control , 3 groups with the different doses of valproic acid sodium ( VPA) and a group with retinoic acid ( RA). VPA and RA were administered producing spina bifida occulta and spina bifida aperta of fetal rat. We analyzed the characteristics of spina bifida induced by VPA and RA in order to study the mechanism. (2) Apoptosis of nerve cells of the fetal spinal cord with spina bifida aperta and protein expression of the correlative genes Bcl-2 and Bax: we detected the nerve cell apoptosis of the fetal spinal cord with spina bifida and the protein expression of the correlative genes Bcl-2 and Bax in order to find the relationship between the apoptosis and spina bifida, and the control of the correlative genes Bcl-2 and Bax on apoptosis with terminal deoxynucleotidyl transferase-mediated dUTP nick end lebeling (TUNEL) and immunohistochemistry methods. So we could investigate further the mechanism of spina bifida. (3)Quantitive analysis of motoneurons innervating the muscle le-vator ani in fetuses with spina bifida occulta: combining the microsurgery on fe-tuses and microinjection technique, we studied the motoneurons by injecting the retrograde tracer fluorogold ( FG) into the muscle levator ani in order to investigating the motoneuron development of the spinal cord with spina bifida occulta. Retrograde tract-tracing technique is commonly used in neuroanatomy.MaterialsAnimals: Wistar rats were provided by Medical Animal Center, the Second Clinical College of China Medical University.Reagents and instruments: valproic acid sodium, retinoic acid, Alcian blue GX, Alizarin red S, fluorogold were provided by Beijing Phetx Co. Ltd. TUNEL kit , immunohistochemistry SABC kit , Bcl-2 and Bax antibodies were provided by Boster Biological Technology Co. Ltd. WuHan. Stereomicroscope and image analysis system are from Olympus Co. Ltd.Methods(1) Animal models : virginal female rats weighting 240 ~ 300g were mated with males of the same strain. On day 9 of gestation, the dose of 20% valproic acid sodium calculated by the weight were injected subcutaneously in the hindquarters at 9 and 16, respectively. On day 20 of gestation, female rats were sacrificed, and the uterus and all its contents removed. Each fetus was inspected for the presence of external malformations and recorded. Then spines of fetuses were double-stained for cartilage and bone using Alcian blue GX and Alizarin red S , and images of double-stained samples were transported into computer by digital camera under stereomicroscope in order to be analyzed by computer software system , which could give the distance parameter of the two cartilaginous ends of the vertebra arch for each vertebra. Compared with the normal fetal distance between the two cartilaginous ends , the spina bifida occulta was diagnosed. On day 10 of gestation, RA was dissolved in mineral oil (40ml/mg) and then administered by gavage feeding (135mg/kg b. w. ) . On day 20 of gestation , Each fetus was inspected for the presence of spina bifida aperta and otherexternal malformations and recorded.(2) Apoptosis of nerve cells of the fetal spinal cord with spina bifida aperta and protein e...