Chemicals and reagents
Rotenone, dimethyl sulfoxide (DMSO), tween80, hydrogen peroxide (H2O2), methanol, phosphate buffered saline (PBS), paraformaldehyde, sucrose, Nissl dye, normal goat serum (NGS), TritonX-100, anti-tyrosine hydroxylase antibody, anti-rabbit IgG (Goat) biotin conjugate, streptavidin, alkaline phosphatase conjugate, and 3-3’-diaminobenzidine were purchased from Chemical Express Co., Ltd., Merck, Millipore, Germany and Agilent, USA. αM and αM-NLC were provided from the National Nanotechnology Centre (NANOTEC) (Pathum Thani, Thailand).
Animals
Forty male ICR mice (Mus musculus) eight weeks old, weighing 40±2 grams, were obtained from the National Laboratory Center, Mahidol University (Nakhon Pathom, Thailand). Mice were housed one per cage at a constant room temperature of 25°C with a 12-hour light/dark cycle. They were fed a standard diet (Mouse diet food NO. 082G) and reverse osmosis (RO) water. The experimental protocol was approved by the Animal Ethics Committee, Faculty of Science, Kasetsart University (ID#ACKU61-Sci-011).
Experimental protocol
Mice were divided into 4 groups as follows: Normal (oral administration of 10% tween80 [vehicle], intraperitoneal injection of 10% DMSO in sunflower oil), PD (oral administration of 10% tween80, intraperitoneal injection of rotenone 2.5 mg/kg dissolved in 10% DMSO in sunflower oil), PD+αM (oral administration αM 25 mg/kg, intraperitoneal injection of rotenone 2.5 mg/kg dissolved in 10% DMSO in sunflower oil), and PD+αM-NLC (oral administration αM-NLC 25 mg/kg, intraperitoneal injection of rotenone 2.5 mg/kg dissolved in 10% DMSO in sunflower oil). All administrations were given every 48 h and continuously for 4 weeks. The dose of αM was selected due to being the lowest effective dose provided from a previous study (Kumar et al. 2016), and the dose of rotenone with exposure time followed the previous protocol that claimed not to have an unspecific effect on body weight and mortality, with benefits for induced PD-like neurodegeneration within 3 weeks (Rahimmi et al. 2015).
Hanging wire test
Muscle strength was assessed by a hanging wire test using a standard wire cage lid. All mice were trained for baseline hanging on the cage lid with the maximum time reached of 120 sec or each mouse have equal hanging ability confirmed by statistical insignificance before the start of the experiment. For testing, the mouse was placed on the top of the lid and allowed to grab the lid with all 4 paws and then turned upside down to start the time of the hanging test. The hanging wire test was performed once a week and consisted of 3 trials with 120 sec, and latency to fall was recorded for muscle strength indication (Yeung et al. 2017).
Rotarod test
Motor coordination was evaluated using a rotarod test (rotary rod diameter 5 cm, length 15 cm, raised 15 cm above the bottom of the rotarod enclosure). The rotarod test was performed once a week. Before the actual test, all mice were trained for 5 consecutive days using a rotation speed starting from 10 rounds per minute (rpm) on the first day and reaching 15 rpm at the end of the training, with 300 sec of maximum time. The rotarod test consisted of 3 trials with a 30-min intertrial time for resting. During the actual test, the animal was placed on a rotating rod, and the speed was gradually increased until it reached 20 rpm. At a fixed speed of 20 rpm, a maximum time of 300 sec was allowed, and the latency to fall was recorded (Zhang et al. 2017).
Biochemical analysis of brain oxidative status
Mice were euthanized with an intraperitoneal injection of sodium pentothal (>60 mg/kg), and quick decapitation was performed. The fresh brains were collected and washed in cold normal saline. They were homogenized in 10% w/v PBS. The homogenates were separated into 2 parts. The 1st part was kept at -20 °C for further evaluation of malondialdehyde (MDA) and reduced glutathione (GSH). The 2nd part was centrifuged at 10,000 g, 4 °C, for 10 min and kept supernatant for further evaluation of total protein, catalase (CAT), and superoxide dismutase (SOD) activities (Sakamula and Thong-Asa 2018).
Nissl staining
Mice were euthanized via intraperitoneal injection of sodium pentothal (>60 mg/kg). Cardiac perfusion with PBS followed by 4% paraformaldehyde was done. Brains were collected and stored in 4% paraformaldehyde at 4 °C for 24 h. After that, brains were processed and embedded in paraffin and sectioned to 5 µm thick with a rotary microtome. Brain sections were cleaned and rehydrated via a serial change of xylene and ethanol 100%, 95%, and 70%, respectively. After 5 min sunk in distilled water, they were stained with Nissl dye (0.1% Cresyl violet) for 30 sec and washed in distilled water, followed by a dehydrating process and finished with cover glass mounted. The images were captured with a light microscope at 200x magnification. The neuronal populations with Nissl staining in SNc, striatum, and motor cortex were evaluated and represented as the percent area of Nissl positive by using NIH Image J (Zhang et al. 2019). The data were presented as the percentage of Nissl positive relative to percentage of control (normal group).
Tyrosine hydroxylase (TH) immunohistochemistry
After being stored at 4 °C in 4% paraformaldehyde for 24 h, brains were moved into 20% sucrose for 48 h at 4 °C. Serial coronal sections (25 µm thick) of striatum (bregma +1.2 mm to +0.25 mm) and SNc (bregma −2.50 mm to −4.24 mm) were cut with cryostat (Churchill et al., 2019). Brain sections were washed in PBS and incubated with 3% H2O2 in 10% methanol followed by incubation with 5% NGS in 0.1% TritonX-100 in PBS. The sections were incubated in 1:200 anti-tyrosine hydroxylase antibody in 2% NGS at 4 °C. After 24 h of incubation, the sections were washed with 0.1% TritonX-100 in PBS and incubated with 1:200 anti-rabbit IgG (Goat) biotin conjugate in 2% NGS for 2 h at room temperature. The sections were washed with 0.1% TritonX-100 in PBS and PBS, respectively, followed by incubation with streptavidin, alkaline phosphatase conjugate in PBS for 2 h, and washing with PBS; then, 3-3’-diaminobenzidine was added for 10-15 min. After washing with PBS, brain sections were mounted with cover glass. The images were captured with a light microscope at 100x magnification, and the percentage of TH intensity was analyzed by using NIH Image J and presented as the percentage of TH intensity relative to percentage of control (normal group).
Statistical analysis
Statistical analyses were carried out using the Statistical Package for Social Sciences version 16.0 (SPSS Inc., Chicago, IL). Data were analyzed by one-way analysis of variance (ANOVA) with Fisher’s PLSD post hoc test. The data were expressed as mean ± standard error of mean (SEM). A value of p < 0.05 was considered statistically significant.