Statistical analyses The statistical analyses were carried out using SPSS version 12.0 for Windows (SPSS, Chicago, IL, USA). As all groups showed normal distribution, intergroup differences were assessed by one- way analysis of variance (ANOVA) and/or Student’s t-test. The results were presented as the means 6 SD. The P value ,0.05 was considered statistically significant. Results Edaravone conspicuously shortened the descent latency in rotenone-treated rats Five weeks after rotenone administration, 22 of 27 (81.48%) Rot- group rats developed characteristic behavioral features of PD, such as back hunching and stiffness, face-washing behaviors, bradykine- sia, or hypokinesia [24]. In these 22 Rot-group rats, 4 rats were fed with Nutrison milk powder 5 g in water twice daily when they stopped eating by themselves, while other 23 Rot-group rats had acceptable health conditions (self-catering, self-cleaning and self- defecation observed). No typical behavioral feature of PD or other health problems were observed in Eda-group and Veh-group (The chemical structure of edaravone was shown in Figure S2). For further accessing the behavioral profile of these animals, prolonged descent latency was found in Rot-group as compared to Eda-group and Veh-group by Grid-iron test (Fig. 1A). Bar Test also showed descent latency in Rot-group was quite longer than Eda-group and Veh-group (Fig. 1B). Edaravone respectively reduced 87.17% and 85.37% the prolongation of descent latency of Grid test and Bar test (Fig. 1). Moreover, there was no significant difference between Veh-group and Eda-group, suggest- ing that edaravone significantly ameliorated the rotenone-induced behavioral defects. Edaravone significantly prohibited rotenone-induced ROS excessiveness in midbrain Flowcytometric data showed higher ROS generation in midbrain of Rot-group than that in Eda-group and Veh-group (Fig. 2). Edaravone inhibited 73.35% of rotenone-induced ROS generation in midbrain tissues. There was no significant difference between Eda-group and Veh-group midbrain tissues in ROS generation, suggesting edaravone is a powerful free radical scavenger. Edaravone protected DA neurons in SNc and DA terminal in CPu Histological examination of brain tissues indicated that rotenone infusion significantly decreased (by 65.67%) integrated intensity of TH immunostaining in CPu (Fig. 3A, C and D), while the TH staining intensity was only decreased by 16.10% in the CPu of Eda-group compared to the CPu of Veh-group (Fig. 4B, C and D). The death rate of TH-positive cells in SNc was 60.48% and 15.82% in Rot- group and Eda-group respectively (Fig. 3E–H). Edaravone rescued 44.66% of dopamine neurons from rotenone-induced degeneration. Moreover, the correlation study on SN degeneration versus behavioral scores indicated that there was a significantly negative relationship between TH-positive cell numbers and the descent latency of Grid test (Fig. 1C) or Bar test (Fig. 1D), although this correlation could be attributed partly to peripheral toxicity as well. Edaravone inhibited apoptotic protein Bax expression in rotenone-treated rats After behavioral test, eight randomly chosen rats of each group were used for immunohistostaining of anti-apoptotic protein Bcl- XL and pro-apoptotic protein Bax. The immunohistological Edaravone Guards Dopamine Neurons examination of the SNc indicated that the rotenone infusion significantly down-regulated the expression Bcl-XL (Fig. 4A) and up-regulated the expression of Bax (Fig. 4D) in the SNc of Rot- group. However, edaravone conspicuously attenuated rotenone- induced down-regulation of Bcl-XL (Fig. 4B). Interestingly, Bax positive cells were not observed in the SNc of Eda-group and Veh- group rats. The quantitative analysis demonstrated that Bcl-XL expression was decreased by 54.36% (Fig. 4A,C,G), as compared to Veh-group. There was no significant difference in Bcl-XL and BAX expression between Eda-group and Veh-group (Fig. 4B,C,E,F), indicating the predominant anti-apoptosis effects of edaravone on rotenone-treated animals. Edaravone ameliorated rotenone-induced mitochondrial damage in the substantia nigra The left and right SNc (from 24.5 to 26.2 mm caudal to the bregma) of the all groups were micro-punched and examined under a transmission electron microscope. There was no ultrastructural difference between the SNc of Eda-group (Fig. 5G,H) and Veh- group (Fig. 5E,F), while mitochondrial swelling, mitochondrial crest fracture, mitochondrial vacuolar degeneration, dilated and broken rough endoplasmic reticula, liberation of ribosomes from rough endoplasmic reticula, the increased formation of primary and secondary lysosomes, and perinuclear space augmentation were observed in SNc neurons of Rot-group (Fig. 5A–C). Medulla sheathes degeneration was also detected in the Rot-group (Fig. 5D), while normal Medulla sheathes were observed in Eda-group (Fig. 5H) and Veh-group (Fig. 5F). Edaravone up-regulated VMAT2 expression without affecting SNCA expression In rat midbrain SNc tissues, edaravone infusion up-regulated the VMAT2 mRNA levels by 45.83% (controlled by GAPDH) and 44.93% (controlled by b-actin, Fig. 6A) in Edaravone-group compared to that in Vehicle-group. Double-immunostaining of TH/VMAT2 showed that VMAT2 protein expression in the DA Figure 2. Effect of Edaravone on Rotenone-induced ROS generation in midbrain. Fluorescent intensity in midbrain of DCF, the product of cellular oxidation of DCFH-DA, as detected by flow cytometry for Rot-, Eda- and Veh-group (*P,0.05 as compared to Rot- group). doi:10.1371/journal.pone.0020677.g002 PLoS ONE | www.plosone.org 4 June 2011 | Volume 6 | Issue 6 | e20677