Experimental Study On Maternal Diabetes Inducing Neural Tube Defect Of Embryo And Its Mechanism

The epidemiological investigation and experiments on animals demonstrate that maternal diabetes or sustained high level of blood glucose could induce embryonic developmental abnormalities,of which neural tube defect(NTD) is frequently observed.However,up to now the mechanism underlying maternal diabetes-induced NTD is not fully understood.In the present study,the mechanism by which the fetal NTD is caused by maternal diabetes has been researched.This study is composed of three portions.Part 1:Establishment of the animal model of maternal diabetes-induced NTD. Diabetes was induced in 8-week-old female Swiss albino mice with streptozotocin (STZ) at a dose of 80mg/kg body weight by intraperitoneal injection on three continuous days.The STZ was dissolved in 0.01M sodium citrate buffer at a concentration of 24mg/ml before use.Blood glucose levels were monitored 3 days after STZ injection using a Glucometer Elite.The mice with non-fasting blood glucose level exceeding 16.6mmol/L were considered as diabetic mice.Diabetic and age-matched control female mice were mated with normal male Swiss mice.Noon on the day that a copulation plug found was considered as embryonic day 0.5.Mice were killed on embryonic day 11.5(E11.5).Embryos were recovered from each pregnancy, All embryos were examined to identify NTD,and then the embryos were fixed in 4% paraformaldehyde(PF) dissolved in phosphate buffered saline(PBS) at 4℃overnight and cryoprotected with 20%sucrose.Transverse sections of 10μm thickness of the developing neural tube were cut using a Leica cryostat.The sections were observed under light microscope for the histological study of NTD.The experimental results show that the rate of NTD or unclosed neural tube is approximately 16%of the total examined embryos,indicating that the animal model of maternal diabetes-induced NTD has been successfully established.Part 2:In vivo study on the mechanism of maternal diabetes-induced NTD.On the basis of the animal model we carried out the in vivo study on the mechanism of maternal diabetes-induced NTD.The cell proliferation and apoptosis of the neuroepithelial stem cells in the neural tube of the embryos from diabetic pregnant mice were investigated by means of BrdU labeling and immunostaining,DAPI staining,TUNEL analysis,statistical analysis etc.The experimental results show that maternal diabetes caused decreased proliferation and increased apoptosis of the neuroepithelial stem cells in the neural tube of the embryos from diabetic pregnant mice,and the difference between the embryos from the diabetic pregnance and the embryos from the normal pregnancy is statistically significant(P＜0.01).Part 3:In vitro study on the mechanism of maternal diabetes-induced NTD: Although the experimental results in part 2 proved that excessive apoptosis and decreased proliferation of the neuroepithelial stem calls were the cellular mechanism by which maternal hyperglycemia induces NTD in embryos from diabetic pregnancy, the results were derived from studying in vivo and complicated by other factors besides blood glucose level.In order to avoid the influence of other factors,the in vitro studies were fulfilled.The neuroepithelial stem cells isolated from the neural tube were cultured in high glucose condition and examined their proliferation and apoptosis by means of several methods.The cell proliferation was examined by BrdU labeling in neuroepithelial stem cells cultured in medium containing respectively 5mmol/L of D-glucose,30mmol/L of D-glucose,5mmol/L of D-glucose plus 25mmol/L of L-glucose.The results show that the percentage of BrdU-positive cells was significantly lower in neuroepithelial stem cells exposed to the 30mmol/L of D-glucose than those exposed to the 5mmol/L D-glucose(P＜0.01).However, exposed to 5mmol/L of D-glucose plus 25mmol/L of L-glucose did not alter the proliferation index of the cultured cells in comparison to that exposed to 5mmol/Lof D-glucose.This finding indicates that it is high glucose,rather than osmotic pressure, that inhibits the proliferation of neuroepithelial stem cell.The cell apoptosis was examined by TUNEL assay in neuroepithelial stem cells cultured in medium containing respectively 5mmol/L of D-glucose,30mmol/L of D-glucose,5mmol/L of D-glucose plus 25 mmol/L of L-glucose.The results show that the percentage of TUNEL-positive cells exposed to 30mmol/L of D-glucose was significantly increased in comparison with those exposed to 5 mmol/L of glucose(P＜0.01),and exposure to 5mmol/L of D-glucose plus 25mmol/L of L-glucose did not significantly alter the cell apoptosis.This finding indicates that high glucose could increase apoptosis of neuroepithelial stem cells,while osmotic pressure has no effect on that..The viability of neuroepithelial stem cells was examined by MTT assay 1 day to 4 days cultured in the medium containing 5mmol/L of D-glucose,30mmol/L of D-glucose,5mmol/L of D-glucose plus 25mmol/L of L-glucose respectively.The results show that high concentration of glucose could significantly inhibit or decrease cell viability of cultured neuroepithelial stem cells,but alteration of osmotic pressure did not influence the cell viability of neuroepithelial stem cell.In addition,the activation of caspase-3 in cultured neuroepithelial stem cells was analyzed by Western blot.The cells were exposed to 5mmol/L and 30mmol/L of D-glucose respectively for 24h,48h,and 72h.The quantity of cleaved caspse-3 in the neuroepithelial stem cells exposed to 30mmol/L of D-glucose was found to be increased compared to those cells exposed to 5mmol/L of D-glucose in a time-dependent manner.