Chemicals and reagents
Betanin, rotenone and other analytic chemicals and reagents were purchased from Chemical Express Co., Ltd., Merck, Millipore, Germany and Agilent, USA.
Forty male ICR mice, 8 weeks old and weighing 30-50 grams, were purchased from the National Laboratory Animal Center (NLAC) at Mahidol University, Salaya, Nakorn Pathom. They were housed in a room with controlled humidity (55%) and temperature (25 oC). They had free access to standard food (No. 082G) and reverse osmosis (RO) water.
The experimental protocol was approved by the Animal Ethics Committee in the Faculty of Science at Kasetsart University (ID#ACKU63-SCI-002). Mice were divided to 4 groups: Sham-veh, Rot-veh, Rot-Bet100 and Rot-Bet200. Mice in the Rot groups received subcutaneous injections of rotenone at 2.5 mg/kg/48h (Rahimmi et al. 2015). Betanin at 100 and 200 mg/kg was dissolved into normal saline (veh was also use as a vehicle) and given via intra-gastric gavage to Bet groups every 48h alternately with rotenone injections. All treatments were given continuously for 6 weeks.
Hanging wire test
A four-limbs hanging test was used to evaluate muscle strength and balance. A cage lid was used as a hanging grid and positioned 25 cm above soft bedding to protect the mouse when it falls. Each mouse was placed on the grid. When it grabbed the grid with four paws, the grid was inverted and the hanging time began. Each mouse had 120 sec maximum with two more tries (three tries total), and the time to fall (sec) was recorded. All mice received training (for base line) before starting the experiment and were tested once a week for 6 weeks (Aartsma-Rus and van Putten 2014).
We used a rotarod test to evaluate fore and hind limb motor coordination and balance impairments (Aartsma-Rus and van Putten 2014). Before the rotenone injection, mice were briefly trained in the rotarod at 11–15 rpm until all mice reach a stable performance (for baseline). A weekly rotarod test was delivered until the end of the experiment. For the test, each mouse was placed on a rotating tube at a steady speed of 5 rpm. The speed was increased from 5 to 15 rpm in 15 sec, and this speed was maintained for at least 180 sec, with two more tries. The running time was collected and expressed as time to fall (sec) for each mouse.
After behavioral tests, mice were euthanized with 180 mg/kg sodium pentothal. They were quickly decapitated and the fresh brains were collected for biochemical analyses. The brains were washed in cold normal saline and homogenized in a 10% w/v phosphate buffer saline (PBS). Half of the homogenate was separated for a malondialdehyde (MDA) assay, and the rest was further centrifuged (10,000g, 4 oC). Supernatant was collected for reduced glutathione (GSH), catalase (CAT) and superoxide dismutase (SOD) assessments (Sakamula and Thong-Asa 2018).
Brains were processed and embedded in paraffin blocks. Five-micrometer sections were created serially using microtomes. The motor cortex (MC) and striatum (Str) sections were collected at bregma -0.34 mm, and SNc sections were collected at bregma -3.52 mm (Paxinos and Franklin 2008). Five sections with 125 µM space intervals were collected from each mouse and stained with 0.1% cresyl violet (Thong-Asa et al. 2020). Brain sections were deparaffined and rehydrated via serial changes of xylene and 100%, 95%, 80% and 70% ethanol. They were dipped in distilled water before staining with 0.1% cresyl violet for 30 sec. They were dehydrated and cleared in reverse via serial changes of ethanol, xylene and finished by sealing with cover glasses. The brain areas of interest (e.g., MC, Str and SNc) were captured via 3 non-overlapping images in each hemisphere. At 100x magnification, two investigators counted viable and degenerating cells in a blind fashion using NIH image J. The data were interpreted as the percentage of degeneration using formula % degeneration = 100 x [degenerating/[viable + degenerating] (Somredngan and Thong-Asa 2018).
Tyrosine hydroxylase (TH) immunohistochemistry
Brains were fixed in cold 4% paraformaldehyde for 24 h (4 oC) and then transferred to PBS containing 20% sucrose. When the brains sunk, they were frozen as 20-µm thick sections with cryostat. Five brain sections covering the Str and SNc area were selected from each mouse with a minimum 100-µm space interval. Brain sections were rinsed in PBS 3x5 min then incubated in 3% H2O2/10% methanol 5-10 min at room temperature (RT). They were washed in PBS for two 5-min intervals and in PBS-T (0.1% Triton X-100 in PBS) for two 5-min intervals and then in the blocking solution (PBS containing 5% goat serum) for 60 min at RT. After a brief rinse in PBS, they were covered by anti-tyrosine hydroxylase (AB152) diluted at 1:500 in carrier solutions (PBS-T containing 2% goat serum). Brain sections were incubated at RT for 3 h before being transferred to a 4 oC environment for a 24 h incubation. The next day, brain sections were rinsed in PBS-T for three 5-min intervals and incubated with biotinylated goat anti-rabbit antibodies diluted at 1:200 (PBS-T containing 2% goat serum) for 60 min at RT. They were washed in PBS-T for 10 min, washed in PBS for two 10-min intervals and then incubated in an ABC solution for 60 min. They were washed in PBS for two 5-min intervals and covered with diaminobenzide for 15 min. After washing in PBS for two 5-min intervals, brain sections were mounted onto cover glasses.
Three non-overlapping images of Str and SNc were capture in each hemisphere. At 100x magnification, TH density was analyzed using NIH Image J and represented as the % TH density related to the % of control (Javed et al. 2016).
Animal weights, latency to falls in hanging wire and rotarod tests, biochemical data and the % of neuron degeneration in MC, Str, SNc and TH optical density related to % of control were analyzed via a one-way variance analysis, followed by a Fisher’s PLSD post hoc test. Statistical significance was accepted for p-values under 0.05.