Figure 4

Autologous EGCs engraft increases neurotrophines release and neurogenesis.

(a) Hippocampal release of NGF, BDNF and GDNF. A time-dependent decrease of neurotrophines was observed in sham compared to vehicle rats (n = 8 and 6, respectively), with the lowest value at 8 weeks (**P < 0.01); in EGCs-transplanted rats the release of neurotrophines progressively increased, being maximum at 8 weeks (°°°P < 0.001; n = 8). (b) Immunofluorescence of BDNF, GDNF and NGF (all in green) in dentate gyrus (DG) of isolated hippocampi; in EGCs-transplanted rats the close contact between PKH26GL-positive EGCs (red) and neurotrophines-expressing fibers (green) is magnified in the squares (blue: DAPI staining of nuclei; magnification: 10 and 25X; scale bar: 100 and 10μm). (c) Quantitative analysis of BDNF, GDNF and NGF showing that Aβ-associated reduction of neurotrophines fluorescence was significantly restored in EGC-transplanted rats (**P < 0.01 vs vehicle and °°°P < 0.001 vs. sham; n = 6 per group). (d) Immunofluorescence of EGC (PKH26Gl, red), Doublecortin X (DCX, green) and DAPI (blue) in DG and subventricular zone (SVZ) showing the reduced neurogenesis in sham compared to vehicle rats (n = 8, respectively). In EGC transplanted rats (n = 8) the increased DCX immunopositivity was evident; a detail of the contact between EGCs and DCX arborizations is magnified in the square (magnification: 10 and 25X; Scale bar: 100 and 10 μm). (e) Quantitative analysis of positive DCX neurons in DG (upper) and SVZ (lower). The amount of DCX neurons was significantly decreased in sham rats, but in EGC-transplanted there was a significant increase in DCX percent (*P < 0.05 and °°°P < 0.001, respectively). Data are representative of mean ± S.E.M of n = 5 experiments in triplicate for each animal group; statistical analysis was performed using ANOVA and Bonferroni’s post-test; values of P < 0.05 were considered significant. (f) Immunofluorescence analysis of NeuN (red) and BrdU (green) in DG. At 8 weeks, BrdU positivity was reduced in sham rats, but it was increased in EGC-transplanted rats (magnification: 10X; scale bar: 100 μm). (g) Quantitative analysis showed that the percentage of NeuN/BrdU co-expressing neurons was significantly reduced in the sham group (*P < 0.05 vs. vehicle); in the transplanted group, NeuN/BrdU immunoreactivity was significantly increased (°°P < 0.01 vs sham), likely suggesting a remarkable rescue of neurogenesis.