Spatial And Temporal Changes Of Palatal Cell Proliferation And Apoptosis In Retinoic Acid Induced Mouse Cleft Palate Varies With The Embryonic Stages

Background: Cleft palate (CP) is one of the most common birth defects in human beings. Its exact mechanism is still not clear because multiple factors are involved. Most cleft palates resulted from the failed fusion of secondary palate shelves, and it is important in shelf growth process for medial edge epithelium (MEE) cells and embryonic palatal mesenchyme (EPM) cells to maintain of the proliferation and apoptosis. Retinoic acid (RA) plays an important role in embryogenesis, by regulating morphogenesis, cell proliferation, differentiation, and extracellular matrix production. The induction of cleft palate by RA varies depending on the different exposed stage of development, but the exact mechanism is not clear yet. BrdU can mix in the S-phase of cell cycle specially and is an ideal parameter to reflect the cell proliferation and to track transplant. TUNEL (terminal-deoxynucleotidyl transferase mediated nick end labeling) is the most popular method to detect apoptosis by detecting DNA degradation in apoptotic cells.Objective: To study the effect of retinoic acid on cell proliferation and apoptosis of palatal shelves by BrdU and TUNEL labeling. Furthermore, analysis of cell proliferation and apoptosis after exposing embryonic mice to RA on GD 10 and GD12 to discover the mechanism of cleft palate.Methods: Female C57BL/6J mice about 10 weeks of age were housed overnight with male C57BL/6J mice and checked for vaginal plugs the next morning, which was designated as day 0 of gestation. Totally 32 pregnant females were obtained. All the pregnant mice were randomly divided intothree groups: GD 10 day RA-treated (10 RA) group (n=16) and GD 12 day RA-treated (12RA) group (n=8),which pregnant mice received by giving a single dose of RA at 100mg/kg body weight at 10 o'clock in morning on GD10 and GD12, respectively. As well as control group (n=8) which pregnant mice received 0.2ml corn oil on GD10. BrdU with 100mg/kg of body weight was injected via abdominal cavity of mice by 20 minutes before all the pregnant mice were killed on GD15. Specimens were prepared for immunohistochemical staining by using BrdU and TUNEL monoclonal antibodies.Result: We observed all immunohistochemical results at the embryonic day 15 with the stage of palatal fusion. 1. The percentage of BrdU positive cells of 10RA embryonic palatal mesenchyme (EPM) is obviously lower than that of control group in both frontal and cross sections (P<0.01). There is no difference between GD12 group and control group (P>0.05). 2. The percentage of BrdU positive cells of 10RA medial edge epithelium (MEE) is obviously lower than that of control group in both frontal and cross sections (P<0.01), and no difference is found between GD12 group and control group (P>0.05). 3. Intense staining of TUNEL is detected in the EPM cells of 10RA groups but not in control group and 12RA group(P<0.01). The percentage of TUNEL positive cells of 12RA EPM is no difference with that of control group(P>0.05). 4. TUNEL staining depicting abundant cell apoptosis in the midline epithelial seam at control group and 12RA group but disappears in the MEE cells at 10RA groups(P<0.01); The percentage of TUNEL positive MEE cells of GD12 group is no difference with that of control group(P>0.05).Conclusion: 1.After exposure of embryonic mice to RA on GD 10 days, abnormal palatal shelves formed in small size, but shelves grow in normal size with failure to fuse after exposure on GD 12. These results suggest that exposure of RA induced different pattern of cleft palate varies with the developmental stages; 2. After exposure of embryonic mice to RA on GD 10, the proliferation and differentiation of palatal mesenchymal cells is inhibited, which contribute to smaller size of shelves and failure to contact;3. The MEE cells keep a bilayer midline epithelial seam after exposure on GD 12 with normally apoptosis happened, indicate that cell apoptosis in MEE cells is not the only process required for palatal fusion.