(A) Neurotrophic effect of VEGF-B
on cultured rat midbrain dopaminergic neurons. Mean count (± s.e.m.) of TH-positive neurons after immunocytochemical staining is plotted. Fluorescent images were acquired digitally on an Olympus IX70 inverted microscope and camera using Olympus MagnaFire software. Morphometric analysis was carried out using Image-J (Wayne Rasband, Bethesda, MD). Antibodies were obtained from Chemicon, Temecula, CA. As compared to untreated control cultures (n = 22 culture dishes) a significant increase in the TH-positive neuron number per culture dish after addition of VEGF-B was apparent. The effect of VEGF-B on TH-positive cell number was concentration dependent. At 0.5 ng/ml (n = 8) no change was noticeable. At 2.5 ng/ml (n = 6) and 5 ng/ml (n = 10) a trend was evident, that reached a statistical significant level at 10 ng/ml (n = 12) and 22.5 ng/ml (n = 11). The mean effect remained the same at the highest tested concentration at 50 ng/ml (n = 4, P = 0.051). Culture dishes were from 5 separate preparations. Data are plotted as mean ± s.e.m. (*P < 0.05; one-way ANOVA followed by a Games-Howell post hoc test to account for small group size and heterogeneity of variance). The statistical significant differences from control are depicted by asterisks. (B) Neuroprotective effect of VEGF-B in a severe rotenone rat midbrain in vitro PD model. As compared to the untreated control cultures (n = 17; black bar) the TH-positive neuron number was reduced after addition of rotenone (40 nM; n = 11; gray bar on the left), this cell loss was rescued by adding 22.5 ng/ml VEGF-B167 (VEGF-B+40 nM rotenone; n = 9; gray patterned bar on the right) prior to rotenone (22.5 ng/ml VEGF-B only cultures; n = 9; black patterned bar). Data are plotted as mean ± s.e.m. (*P < 0.05; one-way ANOVA followed by a Fisher LSD post hoc test). The statistical significant difference from control is depicted by an asterisk. Culture dishes from 5 separate preparations were used.