2.1 Animals and reagents
Specific pathogen-free Sprague-Dawley male rats (age, 6 w; weight, 200–220 g) were purchased from Sibeifu（Beijing, China） Biotechnology Co., Ltd (license No. SCXK2019-0010). The rats were reared in clean cages at 22.0 ± 2.0°C room temperature and 55 ± 10% relative humidity under a 12-hour light and 12-hour dark cycle. The animals were randomly divided into the following three groups: unilateral intrastriatal infusion of 6-OHDA hydrochloride (dissolved in 0.9% normal saline containing 0.02% ascorbic acid) followed by sham rTMS treatment (6-OHDA group), unilateral intrastriatal infusion of 6-OHDA hydrochloride (dissolved in normal saline containing ascorbic acid) followed by rTMS treatment (6-OHDA+rTMS group), and unilateral intrastriatal infusion with normal saline containing ascorbic acid (control group).
2.2 Animal Surgery
Rats in the 6-OHDA group and 6-OHDA+rTMS group were pretreated with desipramine (12.5 mg/kg，Med Chem Express, USA) 30 minutes before the operation. Isoflurane-anaesthetized rats were firmly fixed on a stereotaxic frame (Reward, China). 6-OHDA hydrochloride (20 μg/4 μl，Sigma, USA) dissolved in normal saline containing ascorbic acid（Sigma） was administered into the right striatum at 0.5 μl/min using a 10μl syringe (Hamilton, Switzerland) at the following coordinates: anteroposterior, 0.7 mm; mediolateral, -2.6 mm; dorsoventral, -4.5 mm as previously described [14]. After remaining in place for 5 minutes, the inserted needle was slowly withdrawn (at 1 mm/min). At the 3rd week after 6-OHDA injection, apomorphine (0.5 mg/kg, dissolved in normal saline containing ascorbic acid，Sigma) was administered intraperitoneally [14]. Rats that showed continuous rotation > 2 turns/min towards the contralateral side relative to the injection side after the apomorphine injection were used in further experiments. Rats in the control group were administered 4 μl normal saline containing ascorbic acid into the right striatum using the same method.
2.3 rTMS
The rats were restricted with a plastic holder when receiving rTMS treatment. A round coil (64-mm diameter) (Y064, YIRUIDE, Wuhan, China) was set parallel to the skull of the rats and kept 1 cm away from the rats' head as described previously [7]. The parameters were set as follows: 10 Hz, approximately 1 Tesla magnetic field [7], 1 s stimulation, 9 s interval, 20 min treatment daily for 4 weeks. Rats in the 6-OHDA group were administered the same rTMS protocol, but the coil positioning was perpendicular to the skull of the rats.
2.4 Behavioral tests
The cylinder test, which was used for evaluating forelimb use asymmetry, was performed at the 3rd and 7th weeks after the operation (test 1 and test 2, respectively). The rats were put into a transparent plexiglass cylinder (20 × 30 cm), and a mirror was placed behind the cylinder [15]. The rats were observed for 5 minutes, and the number of times the rat forelimbs contacted the cylinder wall was recorded. The final data were calculated by the equation [(ipsilateral (right) forepaw + 0.5 both paws) / (ipsilateral paw (right) + contralateral paw (left) + both paws)] * 100% [15].
2.5 Immunohistochemistry staining
Glial fibrillary acidic protein (GFAP) and Ionized calcium-binding adapter molecule 1(Iba-1) are generally used as markers of glial cells (astrocytes and microglia). One day after the second behavioral test, 5 rats from each group were selected randomly for immunohistochemistry staining. The anaesthetized animals were intracardially perfused with 0.9% normal saline and 4% paraformaldehyde（Solarbio, China）. The rat brains were rapidly dissected out and immersed in paraformaldehyde at 4 °C. After dehydration and clearing, the tissues were embedded in paraffin and then cut into 5-μm coronal slices. Then, the slices were deparaffinized and hydrated. Afterwards, the slices were unmasked in 0.01 M citric acid buffer and incubated with 3% hydrogen peroxide in the dark followed by primary antibodies (anti-GFAP 1:1000, Abcam; anti-Iba-1 1:1000, Wako, Japan; anti- tyrosine hydroxylase (TH) 1:1000, Millipore, USA) overnight at 4°C. The corresponding secondary antibodies were then added, followed by staining with diaminobenzidine (DAB) until the staining was deemed satisfactory. Hematoxylin staining of the nuclei was carried out, followed by graded ethanol dehydration and fixation in transparent neutral gum. Images were obtained with an Olympus BX53 microscope. ImageJ software was used to analyse positively stained areas.
2.6 Western blot analysis
Rat striatum and SN tissues were lysed in RIPA lysis buffer (Applygen, China) containing protease inhibitor and phosphatase inhibitor (Solarbio) on ice. Protein concentrations in the striatum and SN were determined using a BCA assay (Thermo Fisher Scientific, USA). Protein samples were separated by 10% SDS-PAGE and then transferred to PVDF membranes（Millipore） at 320 mA for 1 h. The membranes were blocked with 5% skim milk for 2 h. The samples were then incubated with the corresponding primary antibodies (anti-HMGB1 1:10000, Abcam, UK; anti-β-actin 1:4000, Proteintech, China; anti-TLR4 1:1000, Santa Cruz, USA; anti- TH 1:1000, Millipore) overnight at 4°C. The next day, the membranes were incubated with the corresponding secondary antibodies (1:10,000，Kerui Biotechnology, China) for 1 h after washing. A chemiluminescence (ECL) reagent (Millipore) were added to visualize the bands after washing. The optical densities of the bands were calculated using ImageJ software.
2.7 Statistics
Statistical analysis was carried out using statistical software (GraphPad Prism 8, GraphPad Software Inc., USA). The apomorphine-induced rotational test of the 6-OHDA group and the 6-OHDA+rTMS group was analysed by t test. The comparisons of other results were analysed by analysis of variance (ANOVA) followed by Sidak's or Tukey's post hoc comparisons. Statistical differences were considered when P < 0.05.