Bacopaside I (BS-I): Phyto-compound was procured from a company Natural Remedies Private limited, Bangalore India and Rotenone (Rot) was obtained from Sigma Aldrich (St. Louis, Mo, USA) for treatment of rats.
Animal experiments:
Wistar strain male albino rats (150-200g, body weight) were used throughout experimental procedure. Experimental animals were obtained with the permission of the Institutional Animal Ethics committee from the breeding colony of Central drug research institute (CDRI), Lucknow. The entire animals were maintained on Hindustan Lever LTD (Mumbai; India) Pellets diet and water ad libitium. Rats were placed under standard conditions of room temperature (22 ± 50C), humidity (45–55%) and light (12/12-h light/dark cycle). Before experimental procedures rats were permitted to acclimate to their new surroundings for 4-6 days. Toxicant rotenone (Rot) was dissolved in 100% dimethyl sulfoxide (DMSO). To stimulate the PD like symptoms in rats, Rotenone (2.0 mg/kg body weight) was administered via subcutaneous injection once daily for 4 weeks. The procedure used here for the induction of Parkinsonism in rats was adopted as study reported earlier (Zhang, Zhang et al. 2017).
For experimental design, rats were divided into three groups with 8 animals in each group as discussed below.
1. Group-I: Subcutaneous injection of DMSO (1 ml/kg body weight) for four weeks.
2. Group-II: Subcutaneously Rot-injected (2mg/kg body weight) for four weeks.
3. Group-III: Received BS-I (dose 5 mg/kg body weight) treatment with oral gavage in addition of Rot injection for four weeks (Zu, Zhang et al. 2017).
4. Group-IV: Received BS-I (dose 15 mg/kg body weight) treatment with oral gavage in addition of Rot injection for four weeks (Zu, Zhang et al. 2017).
5. Group-V: Received BS-I (dose 45 mg/kg body weight) treatment with oral gavage in addition of Rot injection for four weeks (Zu, Zhang et al. 2017).
Total study period for the experimental work was approximately five weeks. After entire dosing, the animals underwent behavioral testing to assess the disease related symptoms.
Neurobehavioral studies:
- Rotarod test
This test was performed by Rotomex (Columbus Instruments, USA) to assess the motor skill coordination in animals. Before, the drug treatment, rats were trained by placing them on the rotating rod (10 rpm) for 300 s. The time of fall from the rotating rod was monitored via following the previous standard procedure (Rai, Yadav et al. 2016).
- Foot printing test
Footprint analysis was used to evaluate the limb coordination. In this test, rats were skilled to walk diagonally on white sheet of paper without stopping. Forepaw of animals were dipped in blank ink and stride length was assessed by measuring space between the centers of ipsilateral adjacent footprint and each step on the same side of the body (Yadav, Prakash et al. 2014).
- Grip strength test
Grip strength test used to determine the measures muscular strength or the maximum force/tension generated by forearm muscles to quantify the effects of toxins or muscle relaxants in the control and treated animals. After placing the animals on grip strength instrument surface, when animal loses its grip, the peak preamplifier automatically stores the peak pull force and shows it on a display board (Terry, Stone et al. 2003).
Brain sample collection:
Animals were sacrificed by cervical dislocation under euthanasia with ketamine hydrochloride. The brain was dissected and frozen immediately. Further whole brain was dissected in ice cold conditions to isolate substantia nigra and striatum region. Brain tissue samples were frizzed at -80 0C till the end of the experiment.
Biochemical Studies:
- ROS Assay
ROS generation was measured based on the oxidation of 2′dichlorodihydrofluorescein diacetate (DCFH-DA) to 2′ 7′ -dichlorodihydrofluorescein diacetate (DCF). Briefly, the tissue homogenate was diluted 1:20 (v:v) with ice-cold Locke’s buffer to achieve a concentration of 10 mg tissue/ml. The reaction mixture (1 ml) was incubated for 45 min at room temperature, and the fluorescence intensity of DCF was measured by excitation at 485 nm and emission at 535 nm using a Microplate Reader. ROS generation was quantified from a DCF-standard curve and data are expressed as pmol DCF formed/min (Chen and Zweier 2014).
- Nitrite assay
Nitrite level was evaluated in the striatum brain tissue homogenate as method previously described (Granger, Taintor et al. 1996). Tissue homogenate and reaction mixtures were incubated at 37 0C for 30 min, and the absorbance of the supernatant was recorded at 540 nm. The level of nitrite was calculated using a standard curve for sodium nitrite (10-100 µM) in terms of µmoles/ml.
- Estimation of dopamine by HPLC
Dopamine assessment was performed by high-performance liquid chromatography (HPLC), In brief, the substantia nigra region of rat brain was homogenized in PBS. The supernatant was collected after centrifugation. The mobile phase consisted of 32 mM citric acid, 12.5 mM disodium hydrogen orthophosphate, 1.4 mM sodium octyl sulfonate, 0.05 mMEDTA and 16% (v/v) methanol (pH 4.2) with a flow rate of 0.8 mL/min. Sample (1 mg/ml) was injected into the chromatographic column. Sample analysis was done at 220 nm wavelength with flow rate (1.0 mL/min) and injection volume (5 μL). Measurements of chromatogram were performed at temperatures of 4°C and 40°C (Yadav, Prakash et al. 2014). The concentration is presented as ng/mg tissue.
Immunohistochemistry:
Tyrosine hydroxylase (TH) immunoreactivity: This anaysis was carried out in the substantia nigra region of brain by immunohistochemical method (Chaturvedi, Agrawal et al. 2003). In brief, animals from each group were deeply anesthetized with sodium pentobarbital (40 mg/kg, i.p.) and perfused transcardially with 0.1 M phosphate-buffered saline (PBS, pH 7.2), followed by 4% paraformaldehyde in PBS for fixation of tissue. Brains were removed immediately and post-fixed in the same fixative for 24 h followed by transfer to 10%, 20%, and 30% sucrose (w/v) in PBS. The fixed tissues were embedded in OCT compound (polyvinyl glycol, polyvinyl alcohol, and water) and frozen at-200C. 20 µm thick coronal sections were cut in freezing microtome (Slee Mainz Co., Germany), and collected in PBS and stored at 40C. Then sections were immersed in wash buffer (sodium phosphate 100 mM, sodium chloride 0.5 M, Triton X-100, sodium azide) pH 7.4 for 20 min. Non-specific binding sites were blocked by incubating the sections in PBS containing 1.5% NGS, 0.5% BSA and 0.1% Triton X-100. These sections were then incubated for 48 h in primary antibody (anti-TH antibody, 1:500). After removing the primary antibody, sections were washed three times with PBS and incubated in Alexaflour 594 secondary antibody (1: 400) for 2 h at room temperature followed by three washes with PBS. Sections were transferred on to gelatinized glass slides, mounted in DAPI, cover slipped and then visualized under microscope.The total number of TH-immunoreactive (TH-ir), neurons in the substantia nigra were determined using a computerized image analysis system (Leica Qwin 500 image analysis software) as described in earlier method (Mochizuki, Yamashita et al. 2001).
Gene expression analysis via quantitative real time q-PCR:
In order to find out gene expression, total RNA was isolated from the hippocampus and substantia nigra region of mice brain using TRIzol Reagent and genomic DNA was removed using RNase-free DNase (Ambion). Pellets were resuspended in DEPC treated water (Ambion). RNA was reverse transcribed through the Superscript first-strand c-DNA synthesis kit with Oligo-dT (Invitrogen, USA). Dilution of c-DNA in nuclease-free water (Ambion) to a final concentration of 10ng/µl. Quantitative real-time PCR was carried out to identify the changes in messenger RNA (mRNA) expression. Expression of the cellular housekeeping gene beta-actin or GAPDH served as a control to normalize values. Targets were detected and quantified in real-time using SYBR Green (Applied Biosystems) in the ABI Prism 7900 HT Sequence Detection System (Applied Biosystems; Foster City, CA) and Relative expression was calculated using the delta Ct method (Tiwari, Agarwal et al. 2015). The sequences for primers are- DAT: F- GTCACCAACGGTGGCATCTA, R- AATTGCTGGACGCCGTAGAA, VMAT: F- TATAACCGCGCAGTCACAGG, R- CTGGGGATGATGGGAACCAC.
Statistical analysis
Statistical differences between different treatment groups were determined by one-way ANOVA with Statistical Package, Graph Pad Prism Version 6.01. All data were expressed as mean ± standard error (SEM) and differences were considered statistically significant, when p values were less than 0.05 (p< 0.05).