Daidzein (96%) was procured from AK Scientific, Inc. USA. Rotenone was obtained from TCI Chemicals Pvt. Ltd. (India). All other chemicals and reagents used in the experiments were of analytical grade. The tyrosine hydroxylase and glial fibrillary acidic protein primary monoclonal mouse antibody belonged to Abcam, (USA). The TNF- α elisa kit was procured from eBioscience, (USA). The reagents for qRT-PCR were procured from DNase, Invitrogen, iScript™ Select cDNA Synthesis Kit, Bio-Rad, SYBR® Green master mix, Bio-Rad.
Animals
The animal study protocol no. ICT/IAEC/2016/P02 was approved by the Institutional Animal Ethical Committee registered under the Committee for the Purpose of Control and Supervision on Experiments on Animals (CPCSEA), India. All the possible precautions were undertaken to minimize the number of animals included in the experiment as their suffering. 5-6 weeks old adult male Sprague Dawley (200-250 g) rats were procured from National Institute of Biosciences (NIBS), Pune-India. These were housed in animal house of Institute of Chemical Technology (ICT) under controlled laboratory conditions of 12 hour light-dark cycle with controlled temperature (25± 2ºC) and relative humidity (50-70%) for at least one week prior to the stereotaxic surgery. All the animals were allowed ad libitum access to food and water.
Experimental design
For the present study, animals were randomly divided as per table 1
No.
|
Group
|
Treatment
|
No. of animals
|
1.
|
Group I
|
Vehicle control (DMSO), intranigral, bilateral
|
10
|
2.
|
Group II
|
Rotenone (12 µg/µl), intranigral (bilateral)
|
10
|
3.
|
Group III
|
Sham control
|
10
|
4.
|
Group IV (DZ-5)
|
Rotenone (12 µg/µl), intranigral (bilateral) +
Daidzein (5 mg/kg) p.o.
|
10
|
5.
|
Group V (DZ-10)
|
Rotenone (12 µg/µl), intranigral (bilateral) +
Daidzein (10 mg/kg) p.o.
|
10
|
6.
|
Group VI (DZ-20)
|
Rotenone (12 µg/µl), intranigral (bilateral) +
Daidzein (20 mg/kg) p.o.
|
10
|
7.
|
Group VII (Standard control)
|
Rotenone (12 µg/µl), intranigral (bilateral)+
Levodopa + Carbidopa (10 mg/kg) p.o.
|
10
|
Table 1: Animal grouping and distribution according to the respective treatment. p.o.- peroral
Stereotaxic surgery and drug treatment:
The animals were prepared for the surgery by induction of anaesthesia with 4% isoflurane, 0.5 L/min O2 inhalational anaesthesia and maintained in it throughout the surgical procedure. Post anaesthesia, the hairs on the surgery area (scalp) were trimmed and the area was disinfected with povidone iodine. The animal was then laid on surgery board with straight head positioned. A midline sagittal incision was made on the scalp and the area below it was cleansed to expose the bregma. The position of Substantia nigra was pinpointed for drilling the burr holes according to the mentioned coordinates Anteroposterior (AP): -5.0 mm from the bregma; Mediolateral (ML): ±2.1 mm from the midline; Dorsoventral (DV): 8.0 mm from the skull. 1 µl Rotenone (12 µg/µl) was infused bilaterally in SN (Paxinos and Watson 2004). The control group animals received DMSO. A sham control group received only burr holes in the skull. Post rotenone administration, the animals was kept on thermal pad, the cut on the scalp was sutured and covered with povidone iodine solution. Each animal was administered gentamicin (40 mg/kg, i.p.) and Tramadol (5mg/kg; i.m.) and returned to their cage for recovery. All animals received a recovery period of one week before the initiation of drug treatment. The treatment duration was of 30 days and each group received their respective doses per day as per the table no. 1. The animals were subjected to neurobehavioural analysis during the study duration which included Rotarod test, Open field test and Barnes Maze test as described in the Fig. 1.
Behavioural analysis
Rotarod test
The rota rod instrument (RR01 Plus, Orchid Scientifics, Nashik, India) was used to evaluate the motor impairment in the rat on 0th, 3rd, 15th and 30th day of the study (Carter et al. 2001). Briefly, the rat was placed on a rotating rod of the instrument at a fixed speed of 30 RPM. The time taken by the rat to fall down from the revolving rod was noted. A predetermined cut off time of 60 seconds (sec) was fixed for the study.
Open field Test
The open field is a square arena made up of plywood with a length and breadth of 72 cm and 36 cm height. Sixteen sub-square divisions of 18 × 18cm were plotted on the floor arena. The test was started by placing the test rat in the centre of the arena and its behaviour was observed for next 5 minutes. After observation the test rat was placed in its home cage and the apparatus was cleaned with 70% ethanol before placing the next test rat. The parameters observed on Day 0, 2 and 29 included number of line crossings (crossing the squares boundaries with both forepaws), number of rears (standing on its hind legs), grooming (rubbing the body with paws or mouth and rubbing the head with paws) and immobility time (Gould et al. 2009).
Barnes Maze Test
The Barnes maze consists of a circular platform with a diameter of 122 cm, consisting of 20 holes evenly punched on the perimeter. A box with its inside painted in black called “an escape box” was attached under one of the holes below the platform. The rat was acclimatised initially by placing it in the centre of the maze into a start tube. After 30 sec, the rat was released from the start tube and the rat was allowed to enter the escape box within 3 min of the cut-off time where it was allowed to stay for 2 min. The first day (acclimatisation phase) was followed by 4 days of acquisition period. The rats were directed to the escape hole during these phases if they failed to locate its position within 3 min. The rats were provided with external cues for better learning. On the last day (probe trial), memory retention was evaluated without the escape box. The “escape latency time” i.e. the time (seconds) required for the rat to find and completely enter the escape box was noted during all the phases. The Barnes maze test was carried on Day 21 to 27 of the study period (Cohen et al. 2013; Li et al. 2013).
Midbrain processing and sample preparation
The rats were euthanized with CO2 asphyxiation after 30 days of study. The midbrains of the rats from both hemispheres were dissected in ice cold conditions. The isolated mid-brains were rinsed in ice-cold isotonic saline for biochemical estimations and separated in to right and left regions. They were snap freezed with liquid nitrogen and stored in deep freezer till further investigations. For biochemical investigations, the mid-brain was homogenised in 10% w/v of ice-cold 0.1 M phosphate buffer saline (pH 7.4). It was centrifuged at 10,000 rpm at a temperature of 4°C for 15 min and used for estimations of biochemical parameters. For immunochemistry analysis, the rats were perfused with 4% paraformaldehyde and the whole brain was stored in it till further analysis.
Biochemical Analysis
Determination of oxidative stress by Superoxide Dismutase (SOD), Catalase (CAT) and Reduced Glutathione (GSH) analysis.
The measurement of SOD, Catalase and Reduced GSH was conducted to assess the oxidative stress in mid-brain due to rotenone administration. The activity of SOD was determined according to the method stated by (Nandi and Chatterjee 1988; Li 2012) with minor modifications. The inhibition of autooxidation of pyrogallol by SOD was measured by noting the rate of change in absorbance at 325 nm for 5 min and presented as unit activity/ mg protein. The Catalase activity was assessed by method described by (Sinha 1972; Aebi 1974) with minor changes. The decomposition of H2O2 was recorded in the terms of rate of decrease in the absorbance at 240 nm for 3 min and the results were expressed as unit activity/ mg protein. The quantification of reduced GSH was carried out according to the protocol described by (Sedlak and Lindsay 1968; Smith et al. 1988) with minor modification. The absorbance of the reaction mixture was read at 412 nm and the quantity was presented as µ mole/ mg protein.
Determination of Lipid Peroxidation (LPO)
The LPO in the mid-brain homogenate was determined by the spectrophotometric assessment of the Thiobarbituric acid reactive substances (TBARS) i.e. the LPO end product malondialdehyde (MDA) (Ohkawa et al. 1979; Reilly and Aust 1999). The absorbance of pink coloured organic layer from the reaction mixture was determined at 532 nm and the LPO was expressed as nmol of MDA/mg of protein.
Determination of protein content
The protein concentration in the mid-brain homogenate was estimated using the Bradford’s method with bovine serum albumin (BSA) standard curve. The colorimetric reaction was measured at 596 nm in a microplate spectrophotometer (Epoch, Biotek, USA) and expressed as mg/ml.
Immunohistochemistry analysis
The immunohistochemical analysis was performed on 4% paraformaldehyde-fixed, 5 µm-thick brain sections fixed on poly-L-lysine coated slide. The sections selected consists of SNpc and striatum. The fixed sections were subjected to through three changes of xylene for 30 min followed by rehydration with absolute alcohol. The endogenous peroxidase activity was quenched by incubating it with 3% hydrogen peroxide in methanol. The slides were further subjected to antigen recovery using citrate buffer at pH 6 for 15 min. After the initial steps the sections were processed separately for Tyrosine Hydroxylase (TH) positive neurons and Glial Fibrillary Acidic Protein (GFAP) detection.
For TH detection, the tissue sections were washed with Tris-buffered saline with Triton-X (TBST) (0.025% Triton X-100) and blocked with 10% normal saline and 1% BSA. The sections were further incubated overnight with primary monoclonal antibody for Tyrosine Hydroxylase (TH) at 4°C. The sections were washed with Tris buffer solution pH 7.4 after incubation and incubated with Poly- Horseradish peroxidase for 30 min. The brown colour change in the tissue was examined after its incubation with the substrate.
For GFAP detection, the antigen recovery was followed with a treatment with Phosphate buffered saline with 0.3 % Triton X-100 (PBST). The tissues were further incubated overnight with the primary monoclonal anti-GFAP antibody (ab10062) at 4°C. After the overnight incubation, the tissues were further incubated with secondary antibody Alexa fluor @ 488 (goat anti-mouse) for 1.5 hrs after a wash with PBS. The GFAP examination was carried out after being counterstained with DAPI (1.5% in PBS) after a wash with PBS (Fukuda et al. 1999; Sakuma et al. 2008).
The coded samples were evaluated for the intensity of staining and number of immune-positive cells under a light microscope at a magnification of 40 X for unbiased assessment.
Determination of TNF-α by ELISA
The TNF-α levels in midbrain was assessed as per the protocol guidelines provided in the ELISA kit by eBiosciences, USA. The TNF-α levels were expressed as pg/mg protein.
Mitochondrial complex I activity estimation
The protocol for mitochondrial complex I inhibition was utilized as described by (Spinazzi et al. 2012) with minor modifications. The mid brain homogenate prepared in sucrose homogenization was used as a source of mitochondria. The activity of NADH: ubiquinone oxidoreductase was analysed by means of a spectrophotometric assay which estimates the complex I activity based on the rate of oxidation of NADH to its non-absorbing oxidized form NAD+ over the time of 4 minutes at 340 nm (the absorption maximum of NADH). The enzyme activity is calculated considering the extinction coefficient of NADH as 6.2 mM− 1 cm− 1.
Mitochondrial biogenesis
The mitochondrial biogenesis potential of the treatment was analysed by determination of the Mitochondrially Encoded Cytochrome C Oxidase I MtCO1/ COX1 target gene relative expression by RT-PCR. For assessment the total RNA from 50-100 mg mid-brain tissue was extracted using guanidinium-phenol-chloroform method. The extracted RNA purity and yield were determined by Nanodrop spectrophotometer (BioTek, USA). The RNA was further subjected to DNase I (Invitrogen, USA) treatment for 1 hr at 37°C and cDNA synthesis was carried out according to the oligo(dT) Bio-Rad kit protocol.
The qRT-PCR was performed in a Bio-Rad CFX96 Real‐Time PCR Machine (Bio-Rad). The method employed Bio-Rad SYBR green master mix (iQSYBR, BioRad). The MtCO1/ COX1 relative expression was determined using gene-specific primer sequence of 5’- CCC CCG CTA TAA CCC AAT ATC AGA C-3’ (forward); 5’- TGG GTG TCC GAA GAA TCA AAA TAG-3’ (reverse) and normalized to 18s rRNA (Sigma) with a primer sequence of 5’-CAT TCG AAC GTC TGC CCT AT-3’(forward); 5’-GTT TCT CAG GCT CCC TCT CC-3’ (reverse). The thermal cycling conditions were set as initial denaturation at 94°C for 3 min; for 35 cycles of 94°C for 30 secs, primer annealing at 60°C for 30 secs. The amplification reaction was run in triplicates with a no-template control and the DNA melt curve analysis was used to confirm product specificity. The relative gene expression was determined using the 2-ΔΔCtmethod for real-time PCR procedures and analysis following the MIQE guidelines (Livak and Schmittgen 2001; Bustin et al. 2009).
Statistical analysis
The results of the present study are represented as mean ± SEM. The data was statistically analysed using GraphPad Prism 5 software (GraphPad Software, San Diego, CA, USA). The analysis was performed by applying one-way ANOVA followed by post-hoc Dunnett’s Test. The Open field test and Barnes maze test were subjected to Two-way ANOVA followed by post hoc Bonferroni Test. The RT-PCR data was subjected to One-way ANOVA followed by post-ANOVA Tuckey’s test. The significance level designated as per the calculations are ### p< 0.001 when compared with Group I i.e. vehicle control group, *, ** and *** when p<0.05, 0.01 and 0.001 respectively when compared with Group II i.e. Rotenone treatment group.