Materials and Methods

Dental pulp tissue extraction and culture: Young rat pups (P5-P6) were anesthetized with sodium pentobarbital or CO2 asphyxiation, decapitated, and the lower jaw was separated from the upper jaw. First and second molars were extracted and the dental pulp was dissected from hard dentin under a dissecting microscope. Dental pulp tissue was then placed in a petri dish containing ice-cold Dulbecco’s modified Eagle’s medium and Ham’s F12 nutrients (DMEM/F12, Invitrogen), 5% fetal bovine serum, L-glutamine (2mM), penicillin (100U/mL), streptomycin (100 µg/ml), amphotericin (2 mg/ml), and gentamicin. Tissue pieces were mechanically dissociated and washed twice with Hank’s balanced salt solution (HBSS) without calcium or magnesium. Tissue fragments were then trypsinized for 25 min with 0.10 µg/ml trypsin and were further mechanically dissociated with fire polished Pasteur pipettes. The tissue was seeded on poly D-Lysine (100µg/ml) treated flasks and chamber slides (Falkon). Cultures were kept in a humidified incubator at 37°C with 95% air and 5% CO2, and the medium was changed every three days. Cells were later subcultured by trypsinization and were used in the experiments after one or two passages.

VMDA tissue extraction and culture: Timed, pregnant Sprague–Dawley rats were obtained from Charles River breeders. The day on which the rats are sperm-positive (a detectable sperm plug) is designated as the first day of gestation (E1). To accurately estimate the age of the embryos, crown-rump length was measured (Olson et al., 1983; Dunnett and Björklund, 1992). Fetuses were removed from pregnant females that had been asphyxiated by CO2. Fetuses were decapitated and brains were dissected. Using a lateral approach, the ventral mesencephalon was located and dissected (Dunnett and Björklund, 1992). VM tissue was then placed in a petri dish containing ice-cold Dulbecco’s modified Eagle’s medium and Ham’s F12 nutrients (DMEM/F12, Invitrogen), 5% fetal bovine serum, L-glutamine (2mM), penicillin (100U/mL), streptomycin (100 µg/ml), amphotericin (2 mg/ml), and gentamicin. Tissue pieces were mechanically dissociated and washed twice with Hank’s balanced salt solution (HBSS) without calcium or magnesium. Tissue fragments were then trypsinized for 10 min with 0.10 µg/ml trypsin and were further mechanically dissociated with fire polished Pasteur pipettes. The tissue was seeded on poly D-Lysine (100µg/ml) treated flasks and chamber slides (Falkon) alone (control) and with previously cultured with dental pulp cells cultured as described above. Cultures were kept in a humidified incubator at 37°C with 95% air and 5% CO2.
Culture treatments with 6-OHDA and cell counting: 6-OHDA (Sigma) was dissolved in water (0.1% Ascorbic Acid). After 24 hours in vitro, co-cultures of dental pulp and VMDA cells and VMDA cells alone were given fresh culture medium (control) or culture media containing 6-OHDA (5µM and 10µM). Toxicity of 6-OHDA and protection by dental pulp cells was determined by counting tyrosine hydroxylase (TH) immunopositive cells. TH+ neurons were counted using a Nikon Eclipse TE300 confocal inverted microscope at 10X magnification.
Statistical analysis: The data were recorded as means +/- standard deviation. Experiments were performed with conditions present in triplicate. ANOVA was the statistical method employed followed by Tukey’s HD post hoc test using Instat 2.03 (Graph Pad Software, San Diego). Significance was set at p<0.05.