Drugs and Chemicals
Buspirone hydrochloride was purchased from Sigma-Aldrich, USA. Fluoxetine was gifted by Intas Pharmaceuticals Pvt. Ltd, India, Urethane was purchased from Hi-media Laboratories Pvt. Ltd, Mumbai, India, Cortisol kit was purchased from Ray Biotech, GA, USA. cDNA Reverse Transcription Kit was procured form Applied Biosystems, USA. All other chemicals were purchased from local distributors.
Approval of experimental protocol
The experimental protocol was approved by the Institutional Animal Ethical Committee (IAEC) of Sinhgad Institute of Pharmacy, Narhe, Pune, constituted as per the Committee for the Purpose of Control and Supervision of Experiments on Animals (CPCSEA). The CPCSEA reg. No of the institute is 1139/PO/a/07/CPCSEA and protocol approval number is SIOP/IAEC/2016/05.Adult male Wistar rats (250-300 g) were procured from the National Toxicology Centre, Pune and were housed in diurnal lighting condition (12h/12h) with a temperature of 25±1°C, relative humidity of 45-55%. Animals had free access to food (Standard chow pellet, Nutrivet Life Sciences, Pune) and water ad libitum.
Induction of social isolation induced anger, aggression and suicidal ideation and treatment schedule
The rats were divided into six groups containing 6 rats each. Group 1- Rats (Normal rats) were not isolated and were kept in their home cage with minimal handling. Group II- Stress control; isolated and administered with distilled water. Group III-Standard group was treated with FLX (30 mg/kg, once a day). Group IV to VI were socially isolated and treated with BUS (10, 20 and 40 mg/kg), animals in Group II to VI were individually isolated in polypropylene cages for 14 days (two weeks) followed by treatment with the respective drugs for next 14 days (2 weeks) i.e., from day 14 to day 28(Wei et al., 2010). At the end of treatments, the following behavior parameters were carried out, followed by non-invasive blood pressure (NIBP) measurement and MRI of the brain. The rats were then sacrificed; hippocampus was isolated and used for estimation of BDNF using PCR.
Assessment of BUS on anger using roller rotation chamber
The in-house roller rotation chamber was developed in the laboratory (Figure 1), the model has been reported previously to measure anger by Awathale et al.,(Awathale et al., 2020) and was used for estimation of anger. Rats were fasted for 24 h and were placed in the Roller rotation chamber. The food was placed in front of rolling wheel in such a way that rat can see/smell food but had no access to it. To obtain food it rolls the wheel in anger and number of wheel rotation signifies anger here. The wheel rotations were recorded using video tracking system (ASTMT2467-SCH80, VJ Instruments, India) for the measurement of parameters like, duration of wheel rolling and number of time's wheel rolled by the animal.
Evaluation of BUS on aggression
Aggression related parameters were measured using the resident intruder paradigm (Wei et al., 2010). The aggressive behavior of the test experimental rats on exposure of naïve rat was video graphed for 10 m for following parameters- Attack bites: Biting to the intruder animal, Wrestling: taking upright posture in order to fight, usually showing by both the animals, Chasing behavior: Pursuit of intruder by test animal, with or without physical contact, Attack latency: Latency time to the first attack (in seconds) from the introduction of intruder animal and Tail rattling: Rapid lateral quivering of the tail just before or after attacking. Total aggression score included addition of attack bites, wrestling, chasing behavior and tail rattling. The attack latency (latency time of the first attack in seconds) from the introduction of the intruder mouse was noted and taken as a measure of impulsivity (Malkesman et al., 2009).
Evaluation of BUS on irritability
The rats were exposed to an uncomfortable stimulus (a puff of air was blown sharply through a straw onto the back of the animal) and observed for its response. Rats which exhibited enhanced reactivity to stimuli were considered to display irritable behavior and their irritability was rated using 6 category scale as follows(Ho et al., 2001, Malkesman et al., 2009).
Score-0: No irritability
1: Startle response (a complicated involuntary reaction) to air puffed on the rat’s back
and to gentle touching of dorsal lumbar region with rod.
2: Biting reaction to gloved hand placed in a cage 1-3 cm in front of the rat snout (Projecting
the nose, jaws, or anterior facial part of animal’s head)
3: Biting reaction to gloved hand pushing the rat backward against the cage wall
4: Resistance to capture by gloved hand
5: Resistance to holding
6: Vocalization during test
Total irritability scores of an individual rat were calculated by adding the individual irritability score of that rat.
Evaluation of BUS on learned helplessness
Learned helplessness was performed immediately after irritability measurement using active avoidance paradigms. The rats were placed in avoidance chamber and underwent 15 attempts of avoidance paradigms of 33/ sec each. In each of 33 second attempt, the first 3 second no shock was given (i.e., the rat could not have escaped to the platform before the onset of shock). If rat did not escape the first 3 seconds, the shock for next 30 seconds was given (0.8 mA intensity). If rat jumped at any time during the attempt, is termed as escape and escape latency was measured. If rats failed to jump from the platform, that was called as “escape failure”. The number of failed attempts of each rat was also measured Evaluation of standardized extract of Centella asciatica leaves on suicidal behavior related traits in laboratory rats.
Evaluation of BUS on OFT parameters
Open field apparatus consisted 25 (5×5) identical squares (20×20 cm). The squares were virtually subdivided into a peripheral and central sector, (9 central squares (3×3)] and the peripheral sector contain the squares close to the surrounded wall (20 cm high). The animals were placed in the central sector and their activity was video-recorded for the 5 m. Locomotor activity was scored when the animal crossed a sector border with both hind-limbs. The peripheral activity, central activity, total activity, rearing and grooming activities were scored.
Evaluation of BUS on locomotor activity
Each animal was observed for a period of 5 m in a square closed field arena (30x30x30 cm) equipped with 6 photocells in the outer wall. Interruption of beam (locomotor exploratory action) was recorded by means of digital counter Actophotometer (INCO, Ambala, India).
Measurement of hemodynamic parameter
Noninvasive blood pressure (BP) parameter in rat was carried out on the 29th day of the experiment. The rat was kept in the restrainer in such a way that the tail of rat remains outside (At most care was taken to avoid stress and struggle in animals). Pressure cuff was applied on the tail and which was further connected to 8 channel data acquisition system PowerLab (ML 870/P, AD Instruments Pvt. Ltd., Australia) (Aswar et al., 2019).
Measurement of size of amygdala using MRI (Magnetic resonance imaging)
The rats were scanned with a small receiver surface antenna and a clinical MRI scanner [1.5 Tesla (T) Siemens].
Assessment of expression of brain derived neurotrophic factor (BDNF) and concentration of serum cortisol level
On the 30th day (to avoid the stress generated if any, during BP measurement) the blood was withdrawn through retro orbital puncture under anesthesia. Blood samples were collected in tubes and centrifuged at 2500 rpm, and serum was separated. Serum was used for cortisol estimation using the 96-well ELISA kit as per manufacturer’s instructions(Bobade et al., 2015).
Quantitative real-time PCR for mRNA measurements
The expression of BDNF mRNA was analyzed using quantitative real-time PCR (qRT-PCR) as previously described with minor modifications (Sagarkar et al., 2017). Briefly, the tissues were homogenized in the Trizol reagent (Ambion, USA), clean-up was carried out using chloroform. The residual DNA was eliminated using a DNA-free™ DNA Removal Kit (Life Technologies, USA). The total RNA reconstituted in nuclease-free water (Invitrogen, USA) was quantified using Biospec Nano Micro-volume UV-Vis Spectrophotometer (Shimadzu, Kyoto, Japan). The quality of the RNA was evaluated by measuring the 260/280 ratio. The 150 ng of total RNA was reverse transcribed using random hexamers and MultiscribeTM reverse transcriptase (Invitrogen, USA) according to the manufacturer's instructions. The PCR conditions used for the reverse transcription were: 25 °C-10 mins; 37 °C-120 mins; and 85 °C-5 min. The cDNA was further subjected to quantitative real-time PCR (qRT-PCR) in StepOneTM RT-PCR System (Applied Biosystems, USA) by using the SYBR green qPCR master mix (Thermo Fisher Scientific, USA) and specific primers for BDNF IX exon and β-actin. The primer sequences used in this study are as follows: BDNF (F-5′- ACCAGGTGAGAAGAGTGATGACCA -3'; R-5' TGGACGTTTGCTTCTTTCATGGGC-3') and β-actin (F-5'- ACTATCGGCAATGAGCGGTTCC-3'; R-5'- CTGTGTTGGCATAGAGGTCTTTACG-3'). Thermal profile used for amplification consisted of three stages: 95 °C-3 mins (1 cycle); 95 °C, 57 °C and 72 °C-30 sec each (40 cycles). All the reactions were performed in triplicates. β-actin was used as a housekeeping gene for the normalization of the data. Fold changes in the BDNF IX mRNA levels were analyzed using 2−ΔΔCT method after normalizing to the b-actin mRNA levels as a housekeeping gene (Schmittgen and Livak, 2008). The results are represented as the fold changes in the mRNA levels (± SEMs).