9cRA reduces 6-OHDA-induced toxicity in ventral mesencephalic cultures. (A) Primary cultures prepared from rat VM (E15) were treated with 6-OHDA (0, 30, 100 μM) for two hours then washed and exposed to vehicle or 9cRA for 22 hours. Cells were fixed and assayed using TH immunostaining. 9cRA (50 nM) alone did not alter the TH (+) neurons in culture; however, it antagonized the 6-OHDA-mediated decrease in TH cell density. (B) A dose-dependent effect of 6-OHDA on loss of TH cells. Treatment with 9cRA (50 nM) significantly antagonized the loss TH cells induced by 6-OHDA (*p<0.05, 2- way ANOVA). The density of TH (+) cells was normalized to the cell density in the non-6-OHDA and non-9cRA (vehicle) -treated group. (C) 9cRA reduced 6-OHDA (100 μM) –mediated TUNEL labeling in VM neuronal culture. (D) Averaged TUNEL density per field. All data were normalized to the mean of TUNEL density in 6-OHDA +veh group. Treatment with 9cRA (50 nM) significantly reduced 6-OHDA- mediated TUNEL activity to control levels (*p<0.001, 1- way ANOVA).