Fig. 3

Evans blue and hPLGF diffusion from the placenta to the fetal brain and effect of in utero PLGF repression on brain VEGF-R1 levels and cortical vasculature. (a, b) Time-course visualization of Evans blue administered by microinjection in the placenta of pregnant mice at GD15. Fluorescence was acquired by transUV illumination (a) and visualized with a false color scale (b). (c, d) Time-course visualization of Evans blue fluorescence in the brain of fetuses after a placental microinjection at GD15. Fluorescence was acquired by transUV illumination (c) and visualized with a false color scale (d). (e, f) Time-course quantification by spectrophotometry of the 595 nm absorbance of the Evans blue signal injected in placentae (e) and the follow-up in the corresponding fetal brains (f). (g) Quantification by ELISA of human PLGF in the brain of fetuses 30 min after injection in the placentas of pregnant mice at GD15. *p < 0.05 vs the control group using the unpaired t test. (h) Microphotograph visualizing eGFP expression 48 h after in utero transfection of placentae from GD15 pregnant mice with an eGFP encoding plasmid. (i, j) triple staining eGFP/Glut-1/Hoechst indicating that eGFP fluorescence (i) is mainly associated with the fetal trophoblastic layer (j) labelled with Glut-1 (arrow heads). Note that the maternal trophoblastic layer which is also labelled by Glut-1 is poorly transfected. The fetal side of the trophoblastic layers is identified by the presence of nucleated red blood cells characteristic of the fetal circulation (arrows). (k) Visualization by Western blot of PlGF, GFP and actin proteins in placentae from non-transfected (sh⁻/GFP⁻), GFP-transfected (sh⁻/GFP⁺) and shPLGF/GFP transfected (sh⁺/GFP⁺) animals. (l, m) Quantification by Western blot of PLGF and GFP expression levels in placentae from non-transfected (sh⁻/GFP⁻), GFP-transfected (sh⁻/GFP⁺) and shPLGF/GFP transfected (sh⁺/GFP⁺) animals four days post-transfection. (n) Quantification by Western blot of VEGF-R1 expression levels in the brain of fetuses from non-transfected (sh⁻/GFP⁻), GFP-transfected (sh⁻/GFP⁺) and shPLGF/GFP-transfected (sh⁺/GFP⁺) placentae four days post-transfection. *p < 0.05 vs the “sh⁻/GFP⁻” group using the one way ANOVA test followed by Tukey’s post hoc test. (o-r) Visualization of the vasculature in the cortex of fetuses from non-transfected (sh⁻/GFP⁻) (o), GFP-transfected (sh⁻/GFP⁺) (p) and shPLGF/GFP-transfected (sh⁺/GFP⁺) (q) placentae. Statistical analysis of vessel disorganization was performed using the χ² test (r)