Ultrastructure of the SNc Rats (7 from ROT-group, 6 from Eda-group and 4 from Veh- group) were deeply anesthetized after behavioral test and perfused through the aorta with ice-cold PBS and then with paraformal- dehyde (4% wt/vol) and glutaraldehyde (1% wt/vol) in PBS [28]. Brains were post-fixed in 2.5% glutaraldehyde at 4uC for 6 h after the separation. A 1-mm3 tissue block from the left and right SNc regions (24.5 to 26.2 mm caudal to the bream) was micro- punched, fixed in PBS containing 2.5% glutaraldehyde, and preserved at 4uC for further processing. The fragments were post- fixed in 1% osmium tetroxide in the same buffer, dehydrated in graded alcohols, embedded in Epon 812, sectioned with an ultramicrotome, and stained with uranyl acetate and lead citrate. The sections were then examined under a transmission electron microscope (TEM; Technai 10, Philips, Netherlands). Pathology of the peripheral organs The pathological evaluation of the peripheral organs was performed on the some rats as immunohistochemistry study (10 from ROT-group, 8 from Eda-group and 6 from Veh-group). The animals were deeply anesthetized and perfused through the aorta with ice-cold PBS and then with paraformaldehyde (4% wt/vol) in PBS. The heart, kidney, liver, lung, spleen, and stomach were taken out and immersed in 4% paraformaldehyde in PBS at 4uC for 24 h before paraffin-embedding. The tissue sections were cut Edaravone Guards Dopamine Neurons into 5-mm pieces, stained with hematoxylin and eosin (HE), and examined under a microscope (B51, Olympus, Tokyo, Japan). Quantification of VMAT2 mRNA levels by qRT-PCR To further study the neuroprotective mechanism of edaravone on cell and rats, four more rats of each group were intraperitoneally injected with edaravone (10 mg/kg, edaravone: DMSO = 9:1, Edaravone-group) or vehicle (PBS: DMSO = 9:1, Vehicle-group) twice in 24 h. Live decapitation was used to sacrifice all the rats, and left and right SNc regions (24.5 to 26.2 mm caudal to the bregma) were micro-punched. The tissues were added into RNase-free 1.5 ml Microfuge tubes with 100 ml TRIzol reagent, and then homogenized in ice by mechanical trituration, followed by additional 900 ml TRIzol reagent added into the tubes for isolating total RNA. Total RNA extraction from brain tissues, reverse transcription and quantitative PCR reactions methods have been described previously [29,30]. Briefly, reverse transcription was performed with Superscript II reverse transcriptase (Qiagen) oligo(dT) as the primer. Quantitative PCR reactions were conducted with SYBR Green PCR Master Mix and primers listed in Figure S1. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and b-actin were used as internal controls. Relative expression levels were calculated by using the 2-DDCT method. VMAT2 and SNCA primers showed amplification efficiency approximately equal (within 5% difference) to that of the internal controls. Figure 1. Catalepsy test of experimental animals (five weeks after the first administration of drugs). (A), (B), descent latency of Rot-, Eda- and Veh-group by Grid test and Bar test, respectively (*P,0.05 as compared to Rot-group); (C), correlation between SN TH-positive cell numbers and Grid test scores; (D), correlation between SN TH-positive cell numbers and Bar test scores. doi:10.1371/journal.pone.0020677.g001 PLoS ONE | www.plosone.org 3 June 2011 | Volume 6 | Issue 6 | e20677