2.1 Experimental animals
Rats (either sex) having 200-250 g of weight attained from the central animal house of Maharaja Ranjit Singh Punjab Technical University, Bathinda, Punjab (India) were used. The experimental animals were kept and handled in accordance with the follow up of Committee for the Purpose of Control and Supervision of Experiments on Animals (CPCSEA) norms, according to which polyacrylic cages were used to accommodate the animals with standard laboratory conditions which include room temperature 22 ± 2 ̊C, relative humidity of 55- 60 % and 12-h light/dark cycle were maintained. Experimental feed pallet and water made available ad libitum. Entire behavioral studies were conducted between 9:00 am to 5:00 pm. The study was approved in two different protocols with reference no. MRSPTU/IAEC/2018/010 (study-1) and MRSPTU/IAEC/2019/09 (study-2) by the Institutional Animal Ethics Committee (IAEC). To avoid variability between experimental groups, aged match animals were used in all experiments for a given treatment.
2.2 Experimental groups
The assigned set animals were divided randomly in the treatment groups. Study-1 contain six groups and study-2 contain five groups, having six animals (n = 6) in each treatment group. (Table 2.1 & 2.2).
2.3 Drugs and chemicals
3-NP was procured from Sigma Aldrich Chemicals Pvt Ltd, Banglore (India); filgrastim (Dr. Reddy's) and Haloperidol (Serenace® Inj., Searle India, and India) were purchased from the local market.
2.4 Treatment schedule
Study 1:Haloperidol induced TD: -Treatment schedule was synchronized in order to evoke TD like feature where the dose of 1 mg/kg Haloperidol administered intraperitoneally (i.p.) daily for 21 days by dissolving in buffered saline of (pH 7.4).Filgrastim was diluted with normal saline (N.S) and administered at a dose of (20, 40 & 60 µg/kg) subcutaneously (s.c.) 6 hours prior to haloperidol treatment. Weekly assessment of behavioral parameters like vacuous chewing movements (VCM), facial jerking (FJ), tongue protrusions (TP), rotarod, and actophotometer were accessed (1st, 7th, 14th, and 21st day) along with their body weights. Striatum and cortex were isolated after sacrifice on day 22 to estimate biochemical parameters include, lipid peroxidation (LPO), reduced glutathione (GSH), nitrite, catalase, protein estimation, and superoxide dismutase (SOD).
Study 2: 3-NP induced HD: -Treatment schedule was planned to evoke HD like feature,in which the dose of 10 mg/kg 3-NP administered intraperitoneally (i.p.) daily for 21 days by dissolving in buffered saline of (pH 7.4). Filgrastim is used similarly to study 1. Weekly assessment of behavioral (rotarod, open field test, narrow beam walk) and biochemical (LPO, GSH, nitrite, catalase, protein, and SOD) parameters were accessed similarly to study 1.
2.5 Measurement of body weight
The percentage change in body weight was calculated on the behalf of the initial and final reading of the body weights, recorded at the 1st and 21st day, respectively before and after haloperidol (study 1) and 3-NP (study 2) treatment (Jamwal & Kumar, 2019).
Change in bodyweight = bodyweight (1st day−21st day) / 1st day body weight × 100.
2.6 Assessment of behavioral activity
2.6.1 Study 1: Haloperidol induced TD
2.6.1.1 Assessment of orofacial movements: On the test day (on 7th, 14th, and 21st day), each rat was gently placed in a small plexiglass cage (30×20×30 cm) for the assessment of oral dyskinesia. Animals were given 10 min to get acclimatized to the observation cage before behavioral assessments. To quantify assessment, the occurrence of oral dyskinesia, hand-operated counters were employed to score facial jerking (FJs), tongue protrusion (TPs), and vacuous chewing movements (VCMs). The behavioral parameters of oral dyskinesia were measured continuously for 10 min. In all the experiments, the scorer was unaware of the treatment given to the animals (Datta et al., 2016).
2.6.1.2 Rotarod test: To evaluate the motor coordination and integrity of the animal rotarod test was performed. The constant speed of 25 rpm was maintained on the apparatus (IMCORP, Ambala, India) having length 30 cm and diameter 7 cm. Fall off latency was measured during the experiment with a cutoff time of 180 (Jamwal et al., 2017).
2.6.1.3 Locomotor activity: The locomotor activity of the experimental rat was monitored using activity meter (IMCORP, Ambala, India). Before subjecting the animal to the cognitive task, they were individually placed in activity meter, and the total activity count (upper beam and lower beam) was registered for 5 min. The locomotor activity was expressed in terms of total photo beams, counts/5 min per animal (Bishnoi et al., 2008).
2.6.2 Study 2: 3-NP induced HD like symptoms
2.6.2.1 Narrow beam walking test: The apparatus comprises of two different platforms (8 cm in diameter) each side of the beam. The beam of 0.5 mm in thickness and 2.0 cm in width and 120 cm in length is placed 1 m above the ground. Sawdust box was placed below the beam to create a cushion effect to fall off the rat. Animals were acclimatized for 5 min. before the training and mean experiment time consist of 120 sec. where the time taken to cross the beam and the number of foot slips has been recorded for each trial. The average of three measured transfer latency yield the final value with the interval of 2 min of inter trial (Jamwal & Kumar, 2016a; Khan et al., 2015).
2.6.2.2 Open Field Test (video tracking system): The behavior of all animals were captured at the end of the study using a video camera (VJ instruments, India) positioned above the square box (open field), comprises of a wooden rectangular black colored apparatus having dimension 61 × 61 × 40 cm. The floor of the apparatus was completely black to avoid any sensitivity issues during tracking. The experimental room was completely black, and an artificial light source was provided to support the video tracking system.To evaluate the locomotor activity, animals were gently placed wooden boxes (OFT), with 36 houses squares. Animals were given 5 min to get acclimatized and allowed to explore the area before behavioral assessments. The exploration in the open field, i.e., ambulation (the number of squares crossed with all paws), distance moved, and time spent moving (active time), time spent without movement (passive time), average speed wasrecorded by a video tracking system (VJ instruments, India) (Aswar et al., 2017). The three times measured activity during the experiment were averaged to produce a final value, and the cut-off time was 10 minutes (fig 2.1). After exposureto area for 10 min, rats were fear conditioned in the area (A square wooden chamber) that were cleaned with 70% ethanol before each session. In all the experiments, the scorer was unaware of the treatment given to the animals (Sivanathan et al., 2015; Zurn et al., 2005). (Fig 2.1)
2.6.2.3 Rotarod test: Similar to study 1.
2.7 Dissection and Homogenization
Animals were sacrificed by cervical dislocation on 22nd day to isolate striatum/cortex for biochemical estimations. Tissue homogenate was prepared in accordance with 10% (wv-1) in 0.1 M phosphate buffer (pH 7.4). The homogenate was centrifuged at 10,000×g for 20 min. The supernatant was separated for biochemical estimation.
2.8 Measurement of Oxidative Stress Parameters
2.8.1 Measurement of lipid peroxidation
In order to make a quantitative estimation of the extent of lipid peroxidation in the striatum and cortex of the brain the method proposed by Wills is used (Wills, 1966). Optical density was recorded 532 nm (Shimadzu spectrophotometer).
2.8.2 Estimation of Nitrite
Colorimetric assay is used for nitrite estimation where nitric oxide is produced in response to nitrite accumulation in the supernatant of striatum which wasestimated by Greiss reagent (0.1 % N-(1-naphthyl) ethylenediamine dihydrochloride, 1 % sulfanilamide, and 2.5 % phosphoric acid) method described as (Green et al., 1982).
2.8.3 Estimation of glutathione levels
To estimate reduced glutathione (GSH)in the brain was estimated using Ellman method, (Ellman, 1959).
2.8.4 Protein estimation
Lowry method was used for the measurement of protein by using Folin phenol as reagent (Gornall et al., 1949).
2.8.5 Catalase activity
Catalase activity was measured according to the method described by (Luck, 1963) and change in absorbance at 240 nm for 2 min interval with 30/60 sec intervals using Shimadzu UV/visible spectrophotometer
2.8.6 Superoxide dismutase (SOD) activity
Superoxide dismutase activity was measured by the proposed method described as (Kono, 1978).
2.9 Statistical analysis
Values are expressed with means ± SD. The behavioral assessment data were analyzed using a two-way analysis of variance (ANOVA) followed by Bonferroni post-hoc test for multiple comparisons. For biochemical parameters, a one-way analysis of variance (ANOVA) followed by Tukey's post-hoc test was used for comparison. p<0.05 was considered statistically significant.