Adult male Wistar rats (Orient Bio Inc., Seongnam, Korea), weighing 250-300 g at the beginning of the experiment, were housed in a room with a 12 hr/12 hr light/dark cycle and free access to food and water. All procedures complied with the guidelines of the institutional animal care and use committee of the Asan Institute for Life Sciences, Seoul, Korea (2011-12-190).

All surgical procedures were performed under general anesthesia induced by intraperitoneal injection of a mixture of 35 mg/kg zoletil (Virbac S.A, Carros, France) and 5 mg/kg rompun (Bayer, Leverkusen, Germany). A total of 11 rats were randomly separated into two groups. The SND group (n=6) received simultaneous injection of QA and 6-OHDA, and the PD group (n=5) received only injection of 6-OHDA. Rats in the SND group received unilateral injections of 15 µg/3 µL QA (Sigma, St. Louis, MO, USA) dissolved in phosphate-buffered saline (pH 7.4) that were targeted at the right striatum (coordinates: anteroposterior [AP], -0.4 mm; lateral [L], 4.0 mm from bregma; ventral [V], -4.0/-5.0 mm from the dura; and AP, +1.2 mm; L, 3.2 mm; V, -3.0/-4.0 mm, with the tooth bar set at -3.2 mm). A total of 40 µg/8 µL QA was delivered into all four sites at a rate of 1 µL/min using a Hamilton syringe (33-gauge) and an automated microsyringe pump (Harvard Apparatus, Holliston, MA, USA). After injection, the needle was kept in place for 3 min to prevent the solution from flowing backward, and was then retracted over the next 5 min. Following the QA injection, 8 µg 6-OHDA (Sigma) in 4 µL of 0.9% saline with 0.1% ascorbic acid was injected into the ipsilateral MFB (coordinates: AP, -2.2 mm; L, 1.5 mm; V, -8.0 mm, with the tooth bar set at +4.5 mm). The PD group received only the unilateral injection of 8 µg/4 µL 6-OHDA (Sigma) that was targeted at the right MFB and administered in the same way as for the SND group.

The stepping test was performed six times: five times without L-dopa (during the intact state, and at 2, 4, 8, and 9 weeks post-surgery) and one time with L-dopa (at 6 weeks post-surgery). The stepping test was performed as previously described (13) with slight modifications. Briefly, both hindlimbs were firmly fixed in one hand of the experimenter, while one of the forelimbs was fixed in the other hand. The test was repeated with both the contralateral and ipsilateral forelimb. The rostral part of the rat was lowered onto a treadmill (Jeung Do Bio & Plant Co., Seoul, Korea) that was moving at rate of 0.9 m/5 sec. The rat's body remained stationary while the unilateral forelimb was allowed to spontaneously touch the moving treadmill track for >5 sec. Stepping tests were performed more than 5 sec with video recording, and the number of steps was counted during initial 5 sec. All of the experiment sessions were video recorded to allow the number of adjusted steps taken in the backward direction to be counted. Every rat performed the stepping test twice in every session, and the number of steps taken was averaged across the two trials. The number of steps taken by the contralateral forelimb was expressed as a percentage of the number taken by the ipsilateral forelimb. At the 6 week time point, 8 mg/kg L-dopa (Sigma) and 15 mg/kg benserazide (Sigma) in 0.9% saline were intraperitoneally administrated 30 min prior to each stepping test.

The apomorphine-induced rotation test was performed at 4 and 8 weeks post-surgery. The rotation test was performed as previously described (14) with slight modifications. Briefly, apomorphine (0.25 mg/kg, Sigma) in sterile water was subcutaneously administrated, and the rat was immediately harnessed to an automated rotometer (Panlab, Barcelona, Spain). Ipsi- and contra-lateral rotations were recorded for 45 min. The data were expressed as the net (contralateral - ipsilateral turns) average rotations per min (RPM). Rats that presented contralateral rotations <6 RPM were regarded as having SND symptoms, and rats that presented contralateral rotations >6 RPM were regarded as having PD symptoms.

All rats were scanned using the microPET Focus 120 system (Siemens Medical Solutions, Inc., Erlangen, Germany) with a 12×12 array of lutetium oxyorthosilicate and 96 detector blocks. All rats were assessed using [¹⁸F]-FDG to investigate metabolic activity and [¹⁸F]-FP-CIT to investigate dopamine transporter function. Scans were performed at 5 and 10 weeks post-surgery. For the [¹⁸F]-FDG PET scan, rats fasted for >12 hr and were then intravenously injected with 1 mCi [¹⁸F]-FDG in 0.2 mL of sterile saline. One hour after injection, the rats were scanned for 30 min under isoflurane anesthesia. For the [¹⁸F]-FP-CIT PET scan, rats were intravenously injected with 1 mCi [¹⁸F]-FP-CIT in 0.4 mL of sterile saline, and then immediately received dynamic scanning for 90 min. The [¹⁸F]-FDG PET data were reconstructed using a 3-dimensional (3D) maximum a posteriori (MAP) algorithm, and the [¹⁸F]-FP-CIT data were reconstructed using a 2D ordered-subset expectation maximization (OSEM 2D) algorithm (15). ASIPro software (Siemens Preclinical Solutions, Knoxville, TN, USA) was utilized to generate a three-dimensional volume of interest that was combined with three regions of interest in different axial sections. The mean uptake was quantified in the ipsilateral and the contralateral hemispheres. The data were expressed as the percentage of ipsilateral mean uptake values against contralateral mean uptake values.

After the final microPET scan, the rats were transcardially perfused with 0.9% saline containing 10,000 IU heparin (Hanlim Pharm, Seoul, Korea), followed by 4% paraformaldehyde in PBS. Brains were extracted and post-fixed overnight in the same fixative, followed by dehydration in 30% sucrose until they sank. Coronal sections (40 µm thick) of the striatum (AP +2.5 to 0.0 mm) and the SN (AP -6.0 to -4.8 mm) were collected using a microtome (Leica, Wetzlar, Germany) and preserved under free-floating conditions in 0.08% sodium azide (Sigma) in PBS at 4℃. For Nissl staining, striatum sections were mounted onto gelatin-coated slides.

Immunohistochemistry was performed as previously described (16). The coronal sections of the SN (rostral section, -4.8 mm; medial section, -5.4 mm; caudal section, -6.0 mm) and striatum were selected and incubated overnight with 1:2,000 mouse anti-TH antibody (Sigma) in 0.5% bovine serum albumin in PBS (pH 7.4). They were subsequently incubated for 2 hr with biotinylated-anti mouse Ig G (1:200; Vector Laboratories, Burlingame, CA, USA) in the same solution. The complexes of primary and secondary antibody were visualized using diaminobenzidine (R&D Systems, MN, USA) colorimeric reaction. For Nissl staining, the striatum sections were rehydrated with an alcohol series of descending concentration, incubated with 0.5% cresyl violet, and dehydrated. Slides were then treated with a clearing agent and coverslipped.

TH-immunopositive cells on both sides of the SN were counted in three coronal sections using a Nikon 80i microscope (Nikon, Tokyo, Japan) with NIS-Elements F3.0 software at 100× magnification. Data were expressed as the percentage of cell loss in the ipsilateral compared to the contralateral SN. The sizes of both the intact and lesioned striatum were analyzed using ImageJ 1.47t (National Institutes of Health, Bethesda, MD, USA). Data were expressed as the percentage of size in the ipsilateral compared to the contralateral striatum.

All data are presented as mean±standard deviation (SD). All statistical analyses were performed using SPSS (version 12.0.1; SPSS Inc., Chicago, IL, USA). Paired t-tests were conducted to analyze differences within groups at different time points in apomorphine test and PET data. Independent samples t-tests were conducted to analyze differences between groups at different time points in stepping, apomophine test and PET data.

All rats showed motor deficit in the contralateral forelimb after lesion surgery. When the stepping test was performed with L-dopa at 6 weeks post-surgery, contralateral paw-touch rate of all rats in the PD group increased, indicating recovery to a level that was almost equal to the intact status, but L-dopa-induced dyskinesias were observed. By contrast, five of the six rats in the SND group did not show an increase in contralateral paw-touch rate or dyskinesias. There was a significant difference between the SND and PD group in the L-dopa response (P=0.003; Fig. 1).

Four of the six rats in the SND group exhibited bilateral touching of the cylinder wall in the apomorphine rotation test performed at 4 weeks post-surgery. The two other rats in the SND group slowly rotated in large, contralateral circles and occasionally exhibited ipsilateral turning behavior. By contrast, all five rats in the PD group showed continuous contralateral rotations. There was a significant difference in RPM between the SND and PD groups at 4 weeks post-surgery (P=0.006; Fig. 2). In the test performed 8 weeks post-surgery, two rats in the SND group showed a slight increase in contralateral rotation but the significant difference in RPM between the two groups remained (P<0.001). In the follow-up test, contralateral rotations of PD group were more increased than those in the first test (P=0.015).

Reconstructed volumes of interest from the [¹⁸F]-FDG PET scans revealed glucose hypometabolism in the lesioned striatum of the SND group at 5 weeks post-surgery (Fig. 3). By contrast, the PD group showed bilaterally symmetrical glucose metabolism at 5 weeks post-surgery (Fig. 3). There was a significant difference in ipsilateral uptake relative to contralateral uptake between the SND group (77.1±7.0%) and the PD group (94.8±6.3%; P=0.003). This difference remained at 10 weeks post-surgery (64.9±11.4% vs. 95.9±3.3%; P=0.003). In both the SND and PD groups, [¹⁸F]-FP-CIT uptake values were lower in the lesioned striatum than in the intact striatum at 5 and 10 weeks post-surgery (Fig. 4). The relative ipsilateral uptake was similar in the SND and PD group at 5 (54.9±16.4% vs. 45.6±6.6%; P=0.246) and 10 (49.6±6.2% vs. 46.8±4.2%; P=0.441) weeks post-surgery.

There was considerable loss of TH-immunoreactive cells in the lesioned SN in both the SND and the PD group. There was no significant difference in cell loss between the groups (74.7±19.4% in SND vs. 92.8±2.7% in PD; P=0.109; Fig. 5). Nissl staining revealed that the SND group had an enlarged ventricle and parenchymal atrophy on the lesioned side, whereas the PD group did not show differences between the two sides of the striatum (Fig. 6). The size of lesioned striatum was reduced by 48.3±12.0% compared to that of intact striatum (Fig. 5, 6).