Figure 4

Exposure to high glucose levels increased the levels of apoptosis in the cranial neural crest cells.

The chick embryos were exposed to simple saline (control) and high glucose (L-Glucose and D-Glucose) until HH10 before being harvested for Pax7 and c-PARP immunofluorescent staining (A–C). (A–C) Immunofluorescent staining against Pax7 was performed in the control and high glucose-treated embryos. The fluorescent images of the transverse sections at the same level focus on the cranial neural crest cells and dorsal-side neural tubes labeled by Pax7 in the control (A), L-Glucose-treated (B) and D-Glucose-treated (C) embryos. (A’–C’) Immunofluorescent staining against c-PARP was performed on the same transverse sections as shown in (A–C), respectively. (A”-C”) The merged images of (A–C) and (A’–C’) added by DAPI staining, respectively. (A”’–C”’) The high magnification images from the sites indicated by the white dotted squares in (A”–C”), respectively. (D) The bar chart showing the comparison of c-PARP positive cranial neural crest cells between the control and high glucose-treated embryos. (E–G) Hoechst33258 (blue) /Propidium iodide (PI) (red) staining was performed in the primary cultures of cranial neural crest cells (from HH9 chick embryos) in the control (E), L-Glucose-treated (F) and D-Glucose-treated (G) CNCCs. Bright blue apoptotic cells were indicated by blue arrows and PI⁺ necrotic cells were indicated by red arrows. (E’–G’) The merged images of F-actin (green) and (E–G), respectively. (H) The bar chart comparing the ratio of apoptosis and necrosis between the control and high glucose-treated embryos. *P < 0.05, **P < 0.01 and ***P < 0.001 indicate significant differences between the experimental and control embryos. Abbreviations: Con, control; L-Glc, L-Glucose; D-Glc, D-Glucose; PI, Propidium iodide; CNCC, cranial neural crest cell. Scale bars = 100 μm in (A–C), (A’–C’), (A”–H”); 50 μm in (A”’–C”’) and 50 μm in (E–G), (E’-G’).