Edaravone was a gift from Simcere pharmaceuticals (Nanjing, China); Bcl-XL and Bax mouse anti-human monoclonal antibody and horseradish peroxidase-conjugated goat anti-mouse IgG secondary antibody were obtained from Santa Cruz (Santa Cruz, CA, USA). Peroxidase-conjugated streptavidin, FITC-conjugated goat-anti-mouse IgG and Cy3-conjugated goat-anti-rabbit IgG were ordered from Millipore (Billerica, MA, USA). TRIzol reagent, Superscript II reverse transcriptase (Qiagen) oligo(dT), SYBR Green PCR Master Mix, Bovine serum albumin, 29,79- dichlorfluorescein-diacetate (DCFH-DA) and collagenase II were from Invitrogen (Carlsbad, CA, USA). Other reagents were of analytical grade and procured locally. Animals Management The experiments described in this paper were approved by the Ethical Committee on Animal Experimentation of Tongji Medical College, Huazhong University of Science and Technology, China (HUST, Approval ID: Y20080256). A total of 65 six-week-old male Wistar rats (200–250 g, from the Center of Experimental Animals, Tongji Medical College, HUST, China) were used. The animals were maintained under standard conditions with 12-hour light/dark cycles and 2262uC and 6065% humidity. They had access to standard laboratory chow and acidified water ad libitum. Rats were randomly assigned to 3 groups: 1) rotenone group (Rot- group), subcutaneous (SC) infusion of rotenone at 2 mg/kg body wt/day (for 5 weeks) mixed with soybean oil and intraperitoneal (IP) administration of 5 ml/kg saline (for 5 weeks, n = 27); 2) edaravone group (Eda-group), SC infusion of rotenone at 2 mg/kg body wt/day (for 5 weeks) mixed with soybean oil and IP administration of edaravone (2 mg/ml in saline) 10 mg/kg body wt/day (for 5 weeks, n = 22); 3) Vehicle group (Veh-group), SC injection soybean oil only in a dose of 1 ml/kg body wt/day (for 5 weeks) and IP administration of 5 ml/kg saline (for 5 weeks, n = 16). For the qRT-PCR study, TH/VMAT2 and TH/SNCA double-staining study, eight more rats of Edaravone-group and eight more rats of Vehicle-group were used. A half of edaravone dose was injected at 7 am and another half at 7 pm. Rotenone, oil and edaravone were injected based on the rat’s updating body weight until they were sacrificed for the experiment. Their body weight was recorded every day to evaluate their health conditions. Ringer lactate 1 ml was injected when they showed sign of dehydration. They were fed with Nutrison milk powder 5 g in water twice daily when they stopped eating by themselves. Specifically, the experimental timeline with number of rats used was shown in Figure S4. Behavioral test (Catalepsy test) The catalepsy set-up consisted of a vertical grid and a horizontal bar to ascertain inert or static behavior. The tests were carried out between 9 am and 3 pm, always in the same context and under standard conditions [16]. All behavioral tests were taken in daylight and rats were trained for 2 days in order to prevent anxiety and unwanted fear. All rats were tested for catalepsy five weeks after rotenone or vehicle infusion. 1) Grid test [16,17,18]: Gridiron of 30 cm wide and 35 cm high with a space of 1.2 cm between each wire was used. Each rat was hung by all four paws on the vertical grid and stopwatch was started as the rat was held on grid. Stopwatch was stopped and time taken by the rats was noted as descent latency. Maximum descent latency time was fixed 180 seconds. 2) Bar test [16,19,20]: rats were placed with both fore paws on a bar, which was 10 cm above the surface in half rearing position. Stopwatch started as the rat was placed on the bar and time noted as the rat removed one paw from the bar. Maximum descent latency time was fixed as 180 seconds. Edaravone Guards Dopamine Neurons Detection of ROS After behavioral test, rats (10 from ROT-group, 8 from Eda- group and 6 from Veh-group) were sacrificed directly and the midbrains were micropunched, intracellular ROS levels were subsequently measured. Formation of ROS was determined after a modification of the procedure described previously [21,22,23]. ROS detection is based on the incubation of the single cells with the nonfluorescent probe DCFH-DA, which diffuses passively through the cellular membrane. With intracellular esterase activity, DCFH-DA forms a nonfluorescent compound DCFH, which can be oxidized to the fluorescent compound DCF by ROS. In brief, small sections of midbrain tissues were micropunched, transferred into separate tubes, and digested with 0.2% collage- nase II for 30 min at room temperature. After carefully removing collagenase II, Buffer solution (2 ml, 0.01 M 0.01 M sterilized phosphate buffered solution (PBS), plus 2 mM EDTA and 0.5% bovine serum albumin) was then added, and filtrated with a microfilter (30 mm). The filtrates were centrifuged (894 g at 4uC for 10 min), the supernatants removed completely, followed by addition of 1 ml of the buffer solution with DCFH-DA (10 mM) and incubation at 37uC for 15 min. All simples were examined for fluorescence expression using flow cytometry (BD, Franklin Lakes, NJ, USA) with an excitation wavelength of 480 nm and emission wavelength at 505–530 nm along with their negative control. Immunohistochemistry staining Brains (10 from ROT-group, 8 from Eda-group and 6 from Veh-group) were removed and placed in 4% paraformaldehyde solution for 24 h. Coronal sections (5-mm) were cut from 24.5 to 26.2 mm caudal to the bregma (SNc) by using a sledge microtome (Wetzlar, Germany) [24]. The sections were de-waxed, hydrated, and the endogenous peroxidase was quenched with 0.3% H2O2 for 30 min. After antigen retrieval, the slides were treated with 0.5% Triton-X100 for 30 min and 5% bovine serum albumin for 30 min. The sections were first incubated overnight with antibody/phosphate buffered solution (PBS; Bcl-xl, or Bax antibody, or tyrosine hydroxylase (TH, mouse monoclonal antibody, Millipore, Billerica, MA, USA) antibody 1:100 dilution) at 4uC and then incubated with FITC-conjugated goat-anti-mouse IgG or the secondary biotinylated goat anti-rabbit IgG or for 60 min and with peroxidase-conjugated streptavidin for 45 min. The immunoreactions were visualized by 3,39-diaminobenzidine for 15–20 min. The pictures were densitometrically observed and analyzed by employing the Image-Pro plus 6.0 software package (Media Cybernetics Inc, Bethesda, MD, USA). For the TH/VMAT2 and TH/SNCA double-staining, 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. Paraformaldehyde perfusion was used to sacrifice all the rats. Embedded with OCT compound, brain tissues were cut into serial 10-mm-thick slices with a cryostat. Immunofluorescent staining was employed to label the TH (Millipore, mouse monoclonal antibody) and VMAT2 (rabbit polyclonal antibody, Sigma-Aldrich), or TH (Millipore, rabbit monoclonal antibody) and SNCA (BD, mouse monoclonal antibody) in the SNc (from 24.5 mm to 26.2 mm caudal to bregma). Expression was visualized by the CY3- conjugated goat-anti-rabbit IgG, FITC-conjugated goat-anti- mouse IgG (Millipore). For counting TH-Positive Cells and densitometrically analyzing VMAT2 expression, a design-based unbiased stereological method and a morphometry / image analysis system (MacBiophotonics ImageJ, McMaster Biopho- tonics Facility, Hamilton, ON, USA) was used as previously described [25,26,27]. PLoS ONE | www.plosone.org 2 June 2011 | Volume 6 | Issue 6 | e20677