Neuroprotective Effects of Delta-9-Tetrahydrocannabinol against FeSO4- and H2O2-Induced Cell Damage on Dopaminergic Neurons in Primary Mesencephalic Cell Culture

Abstract

Delta-9-Tetrahydrocannabinol and other phytocannabinoids have been previously demonstrated to possess neuroprotective effects in murine mesencephalic cell culture models of Parkinson’s disease, in which increased levels of superoxide radicals led to the loss of dopaminergic neurons. In these models, delta-9-tetrahydrocannabinol did not scavenge these radicals but displayed antioxidative capacity by increasing glutathione levels. Based on these findings, in the present study, we investigated whether the neuroprotective effect of delta-9-tetrahydrocannabinol can also be detected in FeSO₄– and H₂O₂-stressed cells. Mesencephalic cultures were concomitantly treated with FeSO₄ (350 μM) or H₂O₂ (150 μM) and delta-9-tetrahydrocannabinol (0.01, 0.1, 1, 10 μM) on the 12^th days in vitro for 48 h. On the 14^th DIV, dopaminergic neurons were stained immunocytochemically by tyrosine hydroxylase, and fluorescently using crystal violet, Hoechst 33342, and JC-1. FeSO₄ and H₂O₂ significantly reduced the number of dopaminergic neurons by 33 and 36%, respectively, and adversely affected the morphology of surviving neurons. Moreover, FeSO₄, but not H₂O₂, significantly decreased the fluorescence intensity of crystal violet and Hoechst 33342, and reduced the red/green ratio of JC-1. Co-treatment with delta-9-tetrahydrocannabinol at the concentrations 0.01 and 0.1 μM significantly rescued dopaminergic neurons in FeSO₄ and H₂O₂-treated cultures by 16 and 30%, respectively. delta-9-Tetrahydrocannabinol treatment also led to a higher fluorescence intensity of crystal violet and Hoechst 33342, and increased the red/green fluorescence ratio of JC-1 when concomitantly administered with FeSO₄ but not H₂O₂. To conclude, delta-9-tetrahydrocannabinol rescues dopaminergic neurons against FeSO₄– and H₂O₂-induced neurotoxicity. Using fluorescence dyes, this effect seems to be mediated partially by restoring mitochondrial integrity and decreasing cell death, particularly in FeSO₄-treated cultures.