Edaravone Guards Dopamine Neurons Figure 5. Ultrastructural changes in SNc. (A): mitochondrial swelling, mitochondrial crest fracture, mitochondrial vacuolar degeneration, dilated and broken rough endoplasmic reticula, liberation of ribosomes from the rough endoplasmic reticula and the increased formation of lysosomes; (B): mitochondrial swelling, mitochondrial crest fracture, dilated and broken rough endoplasmic reticula, perinuclear space augmentation; (C): mitochondrial swelling, mitochondrial crest fracture, the increased formation of secondary lysosome formation;(D): degeneration of the medullary sheathes; (E, G): normal mitochondria, rough endoplasmic reticulum and ribosomes in the SNc neurons of the Veh-group and Eda-group rats; (F, H): normal medullary sheathes from the Veh-group and Eda-group rats (7 rats from ROT-group, 6 rats from Eda-group and 4 rats from Veh-group). doi:10.1371/journal.pone.0020677.g005 Discussion We have shown that the main pharmacologic features of edaravone’s effective neuroprotection include: 1) attenuation of rotenone-induced characteristic parkinsonian behaviors in rats; 2) prevention of rotenone-induced over-generation of ROS in midbrain; 3) inhibition of apoptotic protein Bax expression in the SNc of rotenone-treated rats; 4) prevention of rotenone- induced pathological changes in peripheral organs; 5) protection of mitochondria in SNc neurons against rotenone toxicity; and 6) up- regulation of VMAT2 expression. We have previously reported that both rotenone-based stereo- taxical and systemic parkinsonian rodent models could recapitu- late nigrostriatal DA lesions and mimic the clinical features of idiopathic PD. They both could be used to study pathogenesis, pathology and pathophysiology of PD and to search for effective treatments for PD [24,31,32,33]. However, the stereotaxical model is more suitable for long-term studies as the behavioural changes progress gradually until the 24th week, while the systemic model is better for studies of both nigrostriatal system and peripheral system because of the rotenone-induced peripheral toxicity [24,31,32,33]. In this study, the systemic rotenone-induced parkinsonian rodent model has been chosen for three reasons. 1) Systemic administration is easily exercisable and also highly reproducible (in our hands, 22 of 27 or 81% rats developed typical behavioral features of PD) without going through surgery [24,31,32,33]; 2) this systemic model is capable of reproducing the progression of PD-like pathology while the parkinsonian symptoms could be reversed by L-DOPA [34,35]; 3) this systemic model could be used to examine protective effects in peripheral system as well [24]. Therefore, we postulated that this systemic model helps better understand the protective activity of edaravone. Previous studies have shown that edaravone attenuated neurotoxin-induced decreases in dopamine levels, TH immuno- staining in the SNc [14,15] and CPu [14], indicating neuropro- tective effects of edaravone in PD models. However, edaravone was reported to only reduce MPTP neurotoxicity in SNc but not in CPu in parkinsonian mouse model [15]. Together with our data, the discrepancies of all these results may attribute to: different neurotoxin-based parkinsonian animal models (MPTP, 6- OHDA and rotenone), different administration route (i.p. and i.v.), different dosage of edaravone (1 and 3 mg/kg, 100 and 250 mg/ kg, 10 mg/kg), different animal species (mice versus rat), different characteristics of the behavioral test, alteration of edaravone- activity and affinity over time after lesioning and of different detailed regimen [14]. It has been reported that edaravone exerts its neuroprotection via anti-apoptotic, anti-oxidative and anti-inflammatory pathways in the 6-OHDA-based parkinsonian model, but the detailed anti- apoptotic mechanism has not been explored. Our data have shown that edaravone up-regulated Bcl-XL expression and inhibited Bax expression in parkinsonian rats, helping understand the underlying mechanisms of edaravone’s anti-apoptosis effects, similar to the findings in stroke animal models [36,37]. Other neuroprotective mechanisms may include reduction of Fas- associated death domain protein to suppress apoptotic cell death (in cerebral infarct) [38], alleviation of dysfunction of endoplasmic reticulum with subsequent cell death (as seen in cerebral ischemia) [39], and decrease in MAP kinase activity in astrocytes [40]. The ultrastructural study demonstrated for the first time that edaravone significantly prevented mitochondrial damage and prohibited rotenone-induced typical ultrastructural changes, including mito- chondrial swelling, mitochondrial crest fracture and mitochondrial vacuolar degeneration, in addition to a previous study which PLoS ONE | www.plosone.org 7 June 2011 | Volume 6 | Issue 6 | e20677