A. Photomicrographs of MDA-immunostained sections through the SNpc of mice pretreated with control, 2% creatine, 1% CoQ10 or the combination of two for 7 days followed with a chronic administration of MPTP 40mg/kg daily for 28 days, show an increase in MDA staining in SNpc cells of mice with MPTP and control diet. Creatine, CoQ10 or their combination treatment resulted in a marked reduction of MPTP-induced MDA immunostaining. High magnification inserts show the MDA staining in neurons. B. Using sections from control mice with control diet as the baseline background, stereological analysis showed the total number of MDA-positive neurons in SNpc of MPTP-treated mice was significantly reduced by the treatment of creatine, CoQ10 or their combination. C. Photomicrographs of α-synuclein stained sections through the SNpc of mice described above show a significant increase of α-synuclein accumulation in SNpc of MPTP treated animals on control diet. Creatine, CoQ10 or their combination treatment resulted in a significant reduction of MPTP-induced α-synuclein accumulation in SNpc neurons. High magnification inserts show the α-synuclein staining in neurons. D. Quantification of α-synuclein labeled cells was performed by determining the average number of intensely labeled neurons per section, and only labeled neurons in MPTP-treated groups with staining intensity higher than that of controls were counted. Treatment with creatine, CoQ10 or their combination reduced α-synuclein positive cells in SNpc, with a significant reduction reached by combination treatment. In figure: a, control diet alone; b, control diet with MPTP; c, 2% creatine diet with MPTP; d, 1% CoQ10 with MPTP; e, the combination diet with MPTP. n= 5 mice in each group. Scale bar = 100μm. Data represent means ± SEM, * P < 0.05. E. Confocal photomicrographs of sections through the SNpc of mice on chronic MPTP stained with anti-TH antibody (green) and anti-α-synuclein antibody (red) show the colocalization of the α-synuclein in the TH-positive neurons (yellow in merge), using a Zeiss LSM510 confocal microscope. Scale bar = 100μm.