We have previously demostrated that trophic GDNF treatment reduced ethanol-induced cell death in B-92 cells (ECC- clonal glial cell line). Recently, we also showed an increase of cell death in B-92 cell cultures infected with a replication-defective recombinant adenovirus containing the E. coli lacZ reporter gene (RAd-bgal). In the current study, we have evaluated the neuroprotective effects of adenoviral-mediated GDNF gene therapy on B-92 cells. Briefly, cells were placed on cover slips at a low density (30000 cells each one); 24 hours later they were transduced with either RAd-bgal or Rad-GDNF, an adenoviral vector harboring the rat GDNF gene under the control of the hCMV promoter. Cultures were treated as follows: i) exogenous GDNF was added to noninfected cultures; ii) exogenous GDNF was added to cells infected with RAd-bgal., iii) cultures were incubated with RAd–GDNF. After two days, cultures were fixed and cytochemically processed for X-gal to assess beta-galactosidase activity. At mean time cell death was measured by identification of apoptotic-nuclei with DAPI staining. In each treated group 1500 cells were counted in three separate experiments. The level of significance of differences between treatment was assessed by one-way ANOVA (n = 18, p = 0.0001). ANOVAs were followed by the Tukey’s test. Cultures treated with exogenous GDNF or receiving the GDNF transgene decreased in a highly significant way the level of adenovirus-induced apoptosis. Nevertheless, GDNF treatment could not eliminate basal apoptosis levels in noninfected cells, suggesting the existence of alternative mechanisms of cell death. WE conclude that neuroprotection appears to be determined by the nature of the death inducer.
