Edaravone Guards Dopamine Neurons Figure 6. Edaravone increased mRNA level of VMAT2 in rats SNc. (A) mRNA levels of VMAT2 and SNCA in edaravone-infused SNc tissue compared to Veh-group (* P,0.05 compared to Veh-group); (B) Quantification of VMAT2 immunostaining; (C–F) and (G–J), double-immunostaining of TH/VMAT2 in the SNc of Vehicle rats and edaravone rats, respectively; (K–N) and (O–R), double-immunostaining of TH/SNCA in the SNc of Vehicle rats and edaravone rats, respectively; (Scale bar = 50 mm); (S) Quantification of SNCA immunostaining; (T) Quantification of TH-positive cell numbers. doi:10.1371/journal.pone.0020677.g006 showed edaravone ameliorated mitochondrial respiratory func- tion, restrain, and prevented mitochondrial membrane potential loss induced by MPP+ [41]. More interestingly, we have shown that edaravone is able to up-regulate VMAT2 expression in the related brain region. VMAT2 takes up cytosolic dopamine and reduces the cytotox- icity of dopamine. Deletion of VMAT2 causes lethality in mice [42] and DNA sequence variation in the human gene is associated with PD [43,44]. Consistently, striatal VMAT2 protein levels are reduced significantly in PD patients [45]. Moreover, rotenone inhibits VMAT2 activity and rotenone even induces VMAT2 accumulation of aggregate-like formations consequently to the redistribution of dopamine to the cytosol and apoptosis of DA cells [46]. In addition, up-regulating the expression level of VMAT2 could protect DA cells against neurotoxicity [47,48]. Buildup of cytosolic dopamine is associated with oxyradical stress and possible dopamine interaction with PD-related proteins including SNCA and Parkin [49,50]. Finally, cytosolic dopamine could cause protein degradation abnormality by ubiquitin polymerization and autophagy block, which contributes to protein aggregation in DA neurons [49,51]. Therefore, increased VMAT2 expression may contribute to the neuroprotective effects (Fig. 8). In the present study, the SC rotenone model was a chronic model, and rotenone could accumulate in the SC adipose tissue because of its lipophilic nature [24], together with soybean oil, rotenone slowly entered body fluid circulation. However, edar- avone could rapidly attain the steady-state plasma drug concen- tration through IP injection, which might help to attenuate rotenone-induced toxicity since there is no direct interaction between rotenone and edaravone [52]. Furthermore, the periph- eral organs injury in Rot-group rats could deteriorate their parkinsonian symptoms as the difficulty in eating, self-cleaning and weight losing. These might explain the significant parkinsonian symptoms in Rot-group and effective protection of edaravone on rotenone-induced SNc and peripheral organs. In summary, by regulating multiple molecular and cellular events (Fig. 8), edaravone protects dopamine neurons as well as peripheral tissues very effectively in the rotenone models for PD. The therapeutic potentials of edaravone are supported by not only its antioxidation activity but also gene regulation (Fig. 8). Moreover, it provides anti-apoptotic effects besides anti-oxidative effects in animal models, consistent with its neuroprotection in clinical stroke patients [9,12]. Therefore, edaravone may represent a more plausible medicine for patients with PD than other antioxidants such as vitamin [53]. Since pretreatment of Figure 7. Pathological changes in peripheral organs of Parkinsonian rats. (A): Neutrophilic infiltration in the pulmonary alveoli; (B): neutrophil infiltration in the periportal areas, the expansion of central veins and bile ducts; (C) and (D): Red blood cells were increased in the renal glomeruli and renal medulla; (E): The normal alveolar walls were thin and delicate; (F): the normal hepatic lobules and central veins; (G) and (H): the normal renal glomerulus and renal medulla; (I): hemorrhage and hemosiderin deposition in the spleen of Rot-group rats; (J): the normal white pulp of the spleen; (K) and (L): normal heart and stomach of Rot-group rats (10 rats from ROT-group, 8 rats from Eda-group and 6 rats from Veh-group; Scale bars = 50 mm). doi:10.1371/journal.pone.0020677.g007 PLoS ONE | www.plosone.org 9 June 2011 | Volume 6 | Issue 6 | e20677