Materials
HCQ was purchased from Tehran-Darou company (Tehran, Iran), 6-OHDA (Lot: STBH3207V), apomorphine (Batch: SLBF5500V), and ascorbic acid (Lot: MKCM8021) were purchased from Sigma-Aldrich Company (St. Louic, MO, USA).
Animals and experimental design
Forty-eight adult male Wistar rats, weighing 250±20 g, were used in this study. Animals were obtained from Pasteur Institute (Tehran, Iran) and housed 4 rats per cage under a standard laboratory condition (12 h light/dark cycles starting at 8:00 h, room temperature 23±2°C). The diet and water were freely provided. All the experimental procedures were carried out in accordance with the guidelines of Ethical Committee of Tabriz University of Medical Sciences for Care and Use of Laboratory Animals (IR.TBZMED.VCR.REC.1400.024), and all efforts were made to decrease the number of animals used and minimize the suffering of rats during the study.
Following a week of adaptation to the new conditions, the animals were randomly divided into four groups (n=12) as follows: sham, PD, PD+levodopa, and PD+HCQ groups. Animals in the sham group underwent stereotaxic surgery, and the vehicle of 6-OHDA was injected into the SNpc, and then treated with normal saline (2 ml/kg, p.o.) for 21 days. Animals in the PD, PD+levodopa, and PD+HCQ were received unilateral intra SNpc injection of 6-OHDA. Three weeks following neurotoxin administration, the model was confirmed by behavioral tests. Animals with rotation more than 7 times/min were considered as an established PD model [14]. Then, the PD animals were treated with normal saline (2 ml/kg, p.o.), levodopa (12 mg/kg, p.o., twice daily) [15], and HCQ (100 mg/kg, p.o.) [11], for 21 consecutive days, respectively.
PD model
Animals were anesthetized by injection of ketamine (80 mg/kg, i.p.) and xylazine (8 mg/kg, i.p.), and fixed in the stereotaxic frame symmetrically. 6-OHDA toxin (12.5 µg in 5 µl of a solution containing % 0.2 ascorbic acid) was injected into the left SNpc according to the corresponding aria in Paxinos atlas coordination (bregma: AP=-5.5 mm, DV=-7.3 mm, ML=-2.6 mm) [16]. In the sham group, the same volume of normal saline containing ascorbic acid was injected. Five minutes after the solution infusion (1 µl/min), the needle was removed. Vehicle and toxin were administered slowly at a rate of 1 µl/min using a Hamilton syringe.
Behavioral tests
The behavioral tests, including the Murprogo’s test and apomorphine-induced rotational behavior, were carried out 21 days after 6-OHDA injection, for confirmation of the model, and at the end of the treatments, by a blinded experimenter.
Rotational behavior
Apomorphine-induced rotation is a common test for assessment the extent of motor impairment induced by 6-OHDA. In this study, all animals were tested for rotational behavior 21 days after 6-OHDA injection (before treatment) and 21 days after lesion (after treatment). Animals were individually positioned in a circular cage, and after at least 10 min of habituation, they received a single injection of apomorphine hydrochloride (0.5 mg/kg, s.c.). Following one min of injection, full numbers of rotations were counted for 30 min at 10-min intervals. The number of contralateral turns to the lesion side was considered as positive scores for apomorphine, and the mean rotational numbers were calculated by the positive scores minus the negative scores [17].
Murprogo’s test
To study muscle stiffness, the animals were individually placed on a flat surface. The animal received a score of zero if the animal’s standing and walking were normal. If the animal remained immobile due to muscle stiffness or had difficulty in moving the arms and legs, it received a score of 0.5. Then the right hand of the animals was placed on a wooden platform with 3 cm height; if the animal held its hand on the platform for at least 10 seconds, it received a score of 0.5. This step was repeated for the opposite hand, and if the hand was held steady, they received another 0.5 points. Then, the animal's right hand was placed on a 9 cm platform, and they were given a score of 1 if they held their hand for 10 seconds. This step was repeated for the opposite hand, and if the hand was held steady after 10 seconds, they received another 1 point. The PD animal received a score of 3.5, indicating full rigidity, and the healthy animal (the sham group) received a score of zero [18].
Tissue collection
At the end of the behavioral tests, all rats were anesthetized by a mixture of ketamine (100 mg/kg, i.p) and xylazine (10 mg/kg, i.p) and sacrificed by decapitation. The whole brain was extracted from the skull carefully. For biochemical assessments and Western blotting, brain samples were sliced using a tissue chopper, and the SN was dissected on a cold plate and stored in a -80 °C freezer. For histological examination, the brain was immersed in 4% buffered paraformaldehyde fixation solution for 48 h.
Assessment of oxidative stress status
Brain samples were homogenized in ice-cold 1.15% KCl solution and then centrifuged at 12000 rpm for 15 min at 4 °C to obtain supernatant. The Bradford method was used to estimate protein concentration in the supernatant.
Malondialdehyde (MDA) levels
Thiobarbituric acid reaction (TBAR) colorimetric assay was used to evaluate the concentration of MDA, as a lipid peroxidation marker. Briefly, 200 μl of the supernatant was mixed with 1 ml of thiobarbituric acid, trichloroacetic acid, and 0.9 ml of Tris-HCl (pH 7.4) and incubated for 20 min at 100 °C. Following centrifugation of the samples at 3,000 rpm for 10 min optical density of the supernatant was read at 540 nm in a plate reader, and the results were presented as nmol/mg protein [19].
Glutathione peroxidase (GPx)
Enzyme activity of GPx was assessed spectrophotometrically using the RANSEL kit (Randox Laboratories Ltd.) based on the method of Paglia and Valentine. The absorbance was read at 340 nm at 37°C, and the results were presented as nmol/mg protein.
Total antioxidant capacity (TAC) levels
To determine brain TAC levels, the 2′-azino-bis (3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) method was applied using a Randox total antioxidant status kit (Randox Laboratories Ltd, Crumlin,
United Kingdom). A spectrophotometer was used to measure the absorbance at 600 nm, and the results were expressed as µmol/mg protein.
Western blotting
SN samples were homogenized (1:10, w:v) in 20 mM Tris–HCl (pH 7.4) buffer containing 1 mM NaF, 150 mM NaCl, 1% Triton-100, and protease inhibitor cocktail. The homogenate was centrifuged at 12,000 g for 10 min at 4 °C to obtain supernatant. Protein concentrations were determined in the supernatant using the Bradford method. An equal amount of protein (40 μg) was subjected to electrophoresis on 12.5% SDS-polyacrylamide gel and then transferred onto a PVDF (Roche, UK) membrane. Membranes were blocked in 5% dried milk in 15 mM Tris–HCl (pH 7.4), 150 mM NaCl, and 0.05% Tween 20 (TBST) for 1 h. Subsequently, the membranes were incubated with the primary antibodies (Santa Cruz, CA, USA) against α-synuclein (sc-12767) and β-actin (sc-47778) as an internal loading control overnight at 4 °C. After washing three times with TBST, the membranes were incubated with horseradish peroxidase (HRP)-conjugated goat anti-rabbit IgG secondary antibody (1:5000, sc-2004) for 2 h. Following washing the membranes with PBS, the membranes were placed in enhanced chemiluminescence (ECL) detection solution (Amersham, UK) and exposed to X-ray film to visualize the signals. The density of protein bands was evaluated by Image J software [20].
Histological examination
The blocks of SN were embedded in paraffin, and slides were prepared by dehydration in ascending alcohol series and then cleared in xylene. Subsequently, coronal sections (40 μm) were serially collected (-4.52 mm to -6.04 mm from bregma) from midbrains [21] using a microtome and stained with 0.1% cresyl violet. The representative images of the left hemisphere SNpc were captured, and the numbers of Nissl-stained neurons of SNpc were counted manually under light microscopy (magnification ×200) for three equivalent sections per brain by a blinded person to the study groups.
Statistical analysis
GraphPad Prism 6.01 (GraphPad Software Inc., La Jolla, CA, USA) was used to analyze the data. Values were presented as mean ± S.E.M. One-way ANOVA followed by Tukey post-hoc test was performed for comparison of the differences among the groups. The criterion for statistical significance was set at a p-value <0.05.