| Clinical studies demonstrate that offspring involved in hyperglycemic gestation may have a higher risk of being born with deformities. Neural tube defects(NTDs), is the most common and severe illness of them and can lead to severe malformations. However, there is a lack of wellaccepted and effective animal models to mimic hyperglycemia-induced NTD, and the underlying mechanism of this disease remains inconclusive. This study established a novel hyperglycemiainduced NTD model using chicken embryos. The protective effect of carnosine, an endogenous dipeptide, was also evaluated in this model.First of all, we used chicken embryo to develop a in vivo hyperglycemia induced NTD model and validated this model with various indexes. Various doses of D-glucose(0.025, 0.05, 0.1, 0.2, 0.4 and 0.8 mmol/200 μl/egg) were injected into the air space of the embryo after 24 h, i.e. at Hamburger-Hamilton(HH) stage 7, of incubation to mimic the hyperglycemic environment. After injection, embryos were further incubated to reach embryo development day 5(EDD 5) for analyzing the effect of high-glucose on embryo development. Rates of death, body weight were recorded. The external characterization of neural tube was observed by stereoscopic examination, then the NTD rate(the number of NTD versus all surviving embryos) and somite number were recorded. The internal characterization of neural tube were observed by paraffin section and HE staining. Results showed that high glucose caused increased death and NTD rate and growth retardation, in a dose-dependent manner. High glucose resulted in various patterns of NTDs such as encephalocele, microcephaly, anencephalus, and unclosed neural folds, which were similar to clinical manifestation of NTD. HE staining of the cross section of embryo indicated that high glucose exposure caused an incomplete closure of the dorsal part of the neural tube. The neuroepithelial cells were loosened and disordered; the luminal and basal neural tube surfaces were uneven and the peripheral structures were obscurely formed. To determine these abnormalities were caused by the glucose we administered, the exogenous glucose were radioactively traced using PET/CT-MRI scaning. We found that the glucose was absorbed by embryo and distributed mainly in the CNS of the embryo. Using a glucose assay kit, we detected an increased plasma and brain tissue glucose content, in a dose-dependent manner. The expressions of glucose transporter 1(GLUT1) and 3(GLUT3) genes, which are identified as the major glucose transporters in CNS and facilitate the transport of glucose across the plasma membrane cells were assayed by real-time PCR, and showed a decrease after high-glucose exposure. This down-regulation was probably a result of cellular protective mechanisms under the unfavorable high glucose environment. From within, we chose 0.4 mmol/egg D-glucose as the final dosage for further experiment. In addition, L-glucose was used as an osmotic control because it is not readily utilized by cells. Results showed that L-glucose only caused a quarter of NTDs and less incidence of hypoevolutism when compared to the same dose of D-glucose. This confirmed the leading effect of hyperglycemia and the secondary role of hyperosmolarity on embryonic malformation.In order to prevent the NTD associated with hyperglycemia, the effect of carnosine was studied in this model. Folic acid, which has controversial results on these kinds of NTDs, was also comparatively studied. Carnosine and folic acid were administered into the air space of the eggs at EDD 0(24 h before high glucose exposure) in various doses of 0.1, 0.5, 1, 5 and 10 nmol/egg and then incubated to reach EDD 5. The death and NTD rate, embryo weight were recorded. The internal characterization of neural tube was assayed by HE staining. We found that carnosine has a surprisingly better effect than folic acid in promoting survival and preventing the incidence of NTD. Carnosine at 0.1 and 0.5 nmol/egg(CL and CH) could significantly lower the hyperglycemia-induced NTD rate from 71.9 % to 28.9 % and 18.9% respectively. Folic acid at 1 and 5 nmol/egg(FL and FH) had the similar effects but could only reduce the NTD incidence to 32.2 % and 31.7 %. Carnosine could significantly recover the growth retardation caused by high glucose model, while only a high dose of folic acid had a similar recovering effect as carnosine. HE staining of the cross section of embryos showed that both doses of carnosine could prevent abnormal closure of neural tubes. The morphology of the neural tube in embryos supplied with folic acid was only partly recovered. It was also discovered that a high dose of carnosine could significantly lower the sustained high level of blood glucose, and up-regulate the gene expressions of both GLUT1 and 3, hinting that carnosine might improve the utilization of glucose and the cellular tolerance towards high glucose.The etiology of NTD is involved in genetic factors that regulate important processes during neural tube development and modulate the incidence and severity of the developing phenotype. Pax3 is a paired-domain containing nuclear transcription factor with key roles in neural tube ontogenesis. Therefore, we studied whether Pax3 was involved in mechanisms in the cellular pathology of NTD caused by hyperglycemia. On EDD 2, the expression pattern of Pax3 was observed in chicken embryos after high glucose exposure by IF staining. A strong expression of Pax3 was found in the neuroectodermal cells near the dorsal part of the neural tube in normal chicken embryos and it was drastically diminished after high glucose exposure. Also an asymmetry of bilateral transfer of neuroepithelial stem cells and unclosed neural tubes were found. The supplementation of carnosine before high glucose exposure prevented the loss of Pax3 expression and the abnormal closure of neural tubes. Although folic acid could partly protect the chicken embryo from NTD, the expression of Pax3 was not recovered. In addition, we used methotrexate(MTX) alone as a folate-deficiency NTD inducer instead of glucose, and found that Pax3 expression was unaffected. These proved that the hyperglycemia-induced NTD and downregulation of Pax3 was not related with folic acid deficiency. Furthermore, the expression of Pax3 was detected in EDD 3.5 embryos by western blot and q PCR. The protein expression of Pax3 was very similar with the result of immunofluorescence images. Hyperglycemia caused a downregulation of Pax3 protein expression. Both doses of carnosine could significantly recover Pax3 expression, but folic acid did not improve it. An interesting phenomenon was noticed in the q PCR results. Neither high glucose nor carnosine supplementation influenced the gene expression of Pax3. Also, the expressions of Pax3 downstream genes, including Sox10, NCAM, Shh and c-Met were examined by q PCR. These genes were all suppressed after high glucose exposure, while a high dose of carnosine could significantly restore them.To relate the altered Pax3 protein expression and unaltered gene expression after high glucose exposure, we speculated that hyperglycemia might cause certain modifications on the Pax3 protein. O-Glc NAcylation is a post-translational modification by O-linked β-N-acetylglucosamine(OGlc NAc) moieties at serine or threonine residues of proteins. It is a nutritionally responsive modification, where the concentrations of intracellular glucose is the main regulator. Using western blotting, we found that the overall O-Glc NAc level of EDD 3.5 chicken embryo increased following high glucose treatment. This abnormal level of O-Glc NAc could be significantly prevented by carnosine supplementation, while folic acid showed no effect on O-Glc NAcylation. Since O-Glc NAcylation is mainly regulated by two opposing enzymes, O-Glc NAc transferase(OGT) and O-Glc NAcase(OGA), the gene expression of these two enzymes were determined by real-time PCR. An increased expression of OGT and OGA in EDD 2 hyperglycemic chicken embryos indicated a high level of O-Glc NAcylation was taking place in the developing embryos. The supplementation with high dose carnosine could noticeably down-regulate both enzymes. In addition, the O-Glc NAcylation of Pax3 was examined by co-immunoprecipitation at EDD 3.5. Results showed that the O-Glc NAcylation level of purified Pax3 was significantly elevated in hyperglycemic chicken embryos, while carnosine could prevent the over-activated OGlc NAcylation of Pax3. Confocal scanning analysis was conducted to confirm the OGlc NAcylation of Pax3 in EDD 2 chicken embryos. Result showed that the O-Glc NAc is colocated with the Pax3 protein in the neuroectoderm near the neural tubes of chicken embryos. High glucose caused an increase in O-Glc NAc and a decrease in Pax3 expression. In line with the previous result, carnosine supplementation could significantly prevent excessive hyperglycemiaactivated O-Glc NAcylation and recover the expression of Pax3. Folic acid was still incapable of reversing the abnormal phenomena caused by hyperglycemia.In conclusion, this study has successfully developed a hyperglycemia-induced NTD model in chicken embryos. High glucose conditions induced O-Glc NAcylation of the neural marker Pax3 and was probably the underlying mechanism of this particular NTD. In addition, an association between carnosine and hyperglycemia-induced NTD was discovered, suggesting a potential therapeutic effect of carnosine on birth defects induced by gestational diabetes. The lack of effect from folic acid further proved the specificity of this impaired embryonic neurodevelopment under conditions of metabolic disturbance. Above all, this study provided new insights into how nutrient availability may influence developmental fates of embryos. In addition, it offered a new strategy and revealed the possibility of using endogeneous nutrients for the prevention of metabolism associated embryopathy. |
PDF Full Text Request