Subjects and 6-OHDA-Parkinsonism model
We obtained about 60 adult male mice (30–35 g) from the animal facility of Xuzhou Medical University, and the experimental protocols endorsed by the institutional animal care and use committee. Mice housed in four different cages with the controlled environment: 12/12-h light/dark cycle (lights on at 7 am) and room temperature of 23 ± 1 °C with ad libitum access to food and water.
We used the following materials: 6-OHDA (medchemexpress (china) CAS NO: 28094-15-7), Amodiaquine (medchemexpress (china) CAS NO: 6398-98-7), Primary antibodies: Antityrosine hydroxylase TH-ab112 (Rabbit,1:200 Abcam), Anti Ret [EPR2871] ab134100 (Rabbit,1:1000 Abcam), Beta-actin monoclonal (Mouse,1:5000 Proteintech), AKT mouse monoclonal (Mouse,1:2000 Proteintech), Protein -Glial cell-line derived neurotrophic factor (GDNF)-enQuire BioReagents), For immunohistochemistry (IHC), the ZLI-9018 KIT purchased from ORIGENE was used. Nitrocellulose filter was from ExCell Bio (Excell bio-Shanghai China).
6-OHDA lesion
Animals were anesthetized with sodium pentobarbital [6] and placed in the stereotaxic apparatus with the bite bar set at 0 mm. The skull was exposed, and the burr hole was made using a high-speed dental drill. All animals were administered 6-hydroxydopamine (6-OHDA, Medchemexpress, 10ug in 2 ul of 0.9% saline containing 0.2% ascorbic acid) in the left substantia nigra (3.0 mm posterior to the bregma, 1.3 mm lateral, 4.7 mm ventral to the dural surface) [13]. The sham-operated animals received vehicle only (0.2% ascorbate in 0.9% saline) at the same coordinates. The injection carried out with a 10-ml Hamilton microsyringe, performed over 4 min, and an additional 4 min was allowed before the needle was removed, as described in the previous studies [2, 4].
Elevated body swing test
Body asymmetry conducted, as previously described by Sanberg [40]. Briefly, the animal was first placed into a standard cage on the table for habituation and to attain a neutral position (all four paws on the ground). The animal was held approximately 3 cm from its tail base and elevated above the surface (3 cm) in the vertical axis. A swing recorded whenever the animal moved its head out of the vertical axis to either side and before attempting another rhythm, the animal must return to the vertical position for the next swing to be counted. Oscillations recorded by using a hand counter. The frequency of initial turning of the head or upper body contralateral to the lesioned side was calculated in 20 consecutive trials and normalized, as follows: % contralateral recovery = [1-(lateral turns in 20 trials-10)/10] X 100% [8, 17, 40]. Body asymmetry was assessed at three different times: (i) 14 days after 6-OHDA lesion, (ii) 28 days after 6-OHDA lesion, and (iii) 2 and 4 weeks after treatment. Animals with left side nigra 6-OHDA lesion will exhibit right side biased swings [40].
Apomorphine challenge.
Mice were challenged with apomorphine (0.6 mg/kg, s.c.) at 14 and 28days after 6-OHDA injection, 2 and 4 weeks after treatment. Animals will exhibit swings to the side with more dopaminergic neurons upon apomorphine injection (Sanberg, 1995),[1, 40, 44]. The number of net rotations was evaluated in plastic tubes (19 cm diameter, 22 cm high) in 30 minutes using the video tracking system ANY-maze (Stoelting, Wood Dale, IL, USA) [3, 4]. Four weeks after surgery, animals with an insufficient number of net rotations (< 1 clockwise rotation/min) [35] were discarded (about five mice). Apomorphine challenge was assessed at three different times: (i) 14 days after 6-OHDA lesion, (ii) 28 days after 6-OHDA lesion, and (iii) 2 and 4 weeks after treatment.
Rotarod
The rotarod was performed as described by Jiang et al. [21, 44]. Briefly, the accelerating rotarod apparatus (Insight Scientific Equipments, Ribeirão Preto, SP, Brazil) consists of a grooved metal roller (6 cm in diameter) and separated 9-cm-wide compartments elevated at 16 cm. The spindle speed was increased to 40 rpm over a maximal period of 300 s, and the time spent on the accelerating rotarod and the corresponding rpm were determined. Rotarod performance was assessed at three different times: (i) 14 days after 6-OHDA lesion, (ii) 28 days after 6-OHDA lesion, and (iii) 2 and 4 weeks after treatment.
GDNF, Amodiaquine and combined treatment
In order to reveal the robust effect of the combined therapy, the PD mice model was divided into four groups. The first group was the control, which received vehicle administration; in short, the PD model was injected normal saline (2 ul) through the same coordinates and continuously received an intraperitoneal injection of normal saline (10 ul/g) for 10 days similar to amodiaquine group. The amodiaquine group received a dose as previously described by Kim et al.,[22, 23] briefly; Amodiaquine (medchemexpress-china) CAS NO: 6398-98-7) was dissolved in 0.9% physiological saline at 4 mg/ ml and administered to mice at 40 mg/kg intraperitoneally, for ten days in total at the interval of 24hrs daily. GDNF group received 2 ul of GDNF at the concentration of 4ug/ul [6] through the same coordinates used to administer 6OHDA (3.0 mm posterior to bregma, 1.3 mm lateral and 4.7 mm ventral to the cranial surface). The combined therapy group received both GDNF and amodiaquine (the same protocol).
Weight
Previous studies have reported that one of the factors noticed in the failure of clinical trials was weight loss [5]. The same observation reported in 2019 [47]. The leading cause of weight loss for patients treated with GDNF is elusive. Some studies suggest that intranigrally -administered GDNF may gain access to ventricular spaces and distribute to hypothalamic nuclei, where it alters neurotransmission to affect food intake and eventually may cause weight loss [19]. A similar effect documented by Lapchak, a study that showed that GDNF distributes from lateral ventricle to fourth through the third ventricle and labels the hypothalamus, and this would indicate that GDNF alters hypothalamic neurotransmission which is necessary for feeding behavior [26]. In the current study, we wonder if amodiaquine administration could mimic or counteract the effect of GDNF on weight loss.
Immunohistochemistry
Under sodium pentobarbital anesthesia, mice were transcardially perfused with 100 ml PBS followed by 150 ml of cold 4% paraformaldehyde in 0.1M phosphate buffer (0.1M PB). The brains were removed, fixed for 24 h at 4 ◦C in the same fixative, processed, and embedded in paraffin. Coronal brain sections (5 µm-thick) were cut on a microtome (Leica RM2155, Nussloch, Germany). Parts were deparaffinized in xylene and rehydrated in a gradient of ethanol and distilled water. After being washed three times with 0.01M PBS (for 5 min each), the sections were incubated in H2O2 solution for 5–15 min to block the endogenous peroxidase activity then washed three times with PBS. In order to reduce non-specific staining, the sections were incubated with goat-serum at room temperature for 10-15mins then serum removed without washing. Incubated with the primary antibody in 37*C for 60 min and washed three times with 0.01M PBS, then sections were incubated with a biotinylated goat anti-rabbit IgG. After three piles of washing with 0.01 M PBS, horseradish enzyme-labeled streptomycin was added to react for 10-15mins at room temp. After three times wash with 0.01M PBS, the sections were stained for peroxidase reaction by incubation with a mixture of diaminobenzidine (DAB) for 5–10 min at room temperature. This was followed by recoloration by incubating with hematoxylin for 20seconds, then dehydrated, cleared, and mounted with galvanol and were examined under a light microscope.
Western blot
The proteins of the ventral midbrain were prepared according to the previous studies [14, 28]. In brief, animals (n = 4 per group) were sacrificed, brains rapidly removed, and the ipsi- and contra-lateral ventral midbrain were dissected and immediately put on dry ice for immediate use and in a -80ºC fridge for further use. Tissues were homogenized in RIPA buffer (Beyotime, China) with phosphatase inhibitor and protease inhibitor (Phenylmethylsulfonyl fluoride) at a cocktail of 100: 1 (RIPA: PMSF). Proteins homogenates centrifuged at 4ºC, 15,000 × g for 30 min. The concentrations of proteins determined by the BCA Protein assay kit (Beyotime, China). 20 µg proteins separated by 10% SDS-PAGE and then were electrotransferred (100V, 1 hour) to nitrocellulose membranes. We used 5% skim milk in Tris-buffered saline to block the membrane in 2hour then incubated overnight at 4ºC with one of the following primary antibodies: Antityrosine hydroxylase TH-ab112 (Rabbit,1:200 Abcam), Anti Ret [EPR2871] ab134100 (Rabbit,1:1000 Abcam), Beta-actin monoclonal (Mouse,1:5000 Proteintech), AKT mouse monoclonal (Mouse,1:2000 Proteintech). The next day, membranes incubated with their respective secondary antibodies. After blotting, the bands on the filter were scanned and analyzed with an image analyzer (Lab Works Software UVP upland, CA, USA).
RT-PCR
Total RNA was reverse transcribed into cDNA using PrimeScript™, RT Master Mix [37], and used for quantification of mRNAs encoding TH, DAT, and RET. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA used as an internal control. The PCR program run at 25 °C for 10 min, then 42 °C for 30 min and 85 °C for 5 min [16]. Results were analyzed with a delta-delta Ct method.
Statistical analysis
All data were shown as mean ± SD. Statistical analysis of the results was performed by one-way analysis of variance (ANOVA) followed by Bonferroni post hoc test and the statistical significance level was set at p < 0.05 for all tests.
Liver hepatotoxicity test
Although amodiaquine was reported to halt Parkinson’s disease motor symptoms, we rendered to determine whether the dosage we used is safe to the liver or not. Previous studies reported that AQ is toxic to the liver as it may cause severe idiosyncratic drug reactions (IDR) that included hepatotoxicity and agranulocytosis, necrosis, and presence of inflammatory cells on histopathological analysis [29, 34]. In order to attain this, the liver test was done as described in the protocol (“http://www.bio-protocol.org/e931 Vol 3, Iss 19, Oct 05, 2013). Briefly, blood was collected from the orbital sinus with a microhematocrit blood tube (heparinized). The dropper was used to push out the blood in the heparinized blood tube, and about 300 µl of blood was collected in the 1.5 ml polypropylene test tube. Centrifuged at 1500 x g, 4 °C for 15 min. Cell-free supernatant plasma was carefully taken (about the half volume of blood) and place it in a properly labeled polypropylene test tube and transferred, about 150 µl plasma into the sample cups. The ALT and AST were further determined as in this protocol (“http://www.bio-protocol.org/e931 Vol 3, Iss 19, Oct 05, 2013,” 2013).