Figure 7.

Effects of IGF-1 intracerebroventricular injections on DNA synthesis, S-phase entry, and apoptosis in the cerebral cortex of developing embryos. IGF-1 (30 ng) was injected into the lateral ventricles of E16.5 rat embryos. A, DNA synthesis was assessed in the cerebral cortex at 6 and 12 h after IGF-1 injection, using percentage [³H]thymidine incorporation assay. [³H]Thymidine was injected subcutaneously into pregnant dams 1 h before the killing. IGF-1 elicited a 12% increase in [³H]thymidine incorporation at 6 h (PBS-treated animals, n = 29; IGF-1-treated animals, n = 30; 5 pregnant dams) and 26% increase at 12 h (PBS-treated animals, n = 20; IGF-1-treated animals, n = 17; 4 pregnant dams) compared with the control group. B, C, Analysis of apoptosis 12 h after injection using activated caspase-3 as a marker. B, Rare cleaved caspase-3-labeled cells (arrows), exhibiting cytoplasmic signal, were revealed in the proliferating and postmitotic cortical zones of both PBS- and IGF-1-injected embryos. C, Western blotting analyses performed on the cortices of injected embryos revealed two bands (17 and 19 kDa) corresponding to the large fragments of activated caspase-3. No difference in protein levels assessed by densitometry was observed between control and IGF-1 treated animals (p > 0.05). Data are representative of three experiments, four animals per group. D, Analysis of cells in S-phase using BrdU immunohistochemistry. BrdU was injected into pregnant dams 1 h before the killing. BrdU-labeled cells were counted over total cells in the VZ of the dorsolateral cortex. IGF-1 intracerebroventricular injection elicited a 26% increase in the BrdU LI at 12 h. Data are from four animals per group. LV, Lateral ventricle. Values shown represent the mean ± SEM. *p < 0.01; **p < 0.001.