Experimental animals
Forty-eight Swiss albino mice (24 male and 24 female adults) of weight range 20-25 g were received from the CRI, Kasauli and LUVAS, Hisar after approval of the protocol no. ASCB/IAEC/08/15/108 (dated: 28-11-2015) by the Institutional Animal Ethics Committee (IAEC) at A.S.B.A.S.J.S.M. College of Pharmacy, Ropar, India. Animals were reserved in isolation till confirmation of their health status and then relocated to the housing section. Three mice of the same sex were kept in a single polyacrylic cage (30 × 23 × 14 cm3) under typical laboratory situations with controlled humidity (40 ± 10%), temperature (23 ± 2℃), and light-dark cycle (12 h each). Rodents were provided two weeks for customization to the housing/laboratory environment prior to the experiments. Guiding principles of “The Committee for the Purpose of Control and Supervision of Experiments on Animals (CPCSEA)” GOI were pursued for the entire course of animal experiments and care of animals. Unrestricted access to typical rodent pellet diet (Ashirwad Industries, Mohali) and purified water was provided to the mice. Caretakers were blinded to treatments that the animals received. All the experiments and procedures were conducted within 12 h cycle (0800 to 2000 h).
Drug treatments
(−)-Nicotine hydrogen tartrate [(−)-1-Methyl-2-(3-pyridyl)pyrrolidine (+)-bitartrate salt] (Sigma-Aldrich, Bengaluru, India) was prepared freshly (dose 3.35 mg/kg) (Biala et al. 2014) using sterile 0.9% normal saline (pH 7.3, dose volume of 2 ml/kg). (−)-nicotine hydrogen tartrate (Nic) was administered subcutaneously (s.c.) from day 1 to 7 thrice a day (t.i.d.) at 5 h intervals (0900, 1400, and 1900 h) (Matta et al. 2007). The intraperitoneal (i.p.) doses of lacidipine (LCD), sertraline, and Bay-K8644 were constituted using a 0.2% dimethyl sulfoxide (DMSO) vehicle. Lacidipine (TCI Chemicals, Chennai, India) at doses 0.3, 1, and 3 mg/kg (i.p.) was injected at a dose-volume of 3 ml/kg once daily for 14 consecutive days initiating from day 1 (Khurana and Bansal 2019a; Bellosta et al. 2001). Sertraline (Zee Laboratories, Ponta Sahib, India), a selective serotonin reuptake inhibitor (SSRI), was taken as a standard drug in the current protocol and was administered (40 mg/kg, i.p.) in mice at dose-volume 4 ml/kg for 14 days once daily (Noori et al. 2014). LCD and sertraline were injected 60 min before Nic administration. Bay-K8644 (Sigma-Aldrich, Bengaluru, India) is an LTCC agonist and was given at a dose 0.5 mg/kg (dose-volume 2 ml/kg, i.p.) 120 min before behavioral tests on day 13 and 14 (Khurana and Bansal 2019b; Jackson and Damaj 2009). The whole dose-volume was reserved ≤ 10 ml/kg body weight (b.w.) per day in each mouse.
Experimental protocol
Male and female mice were randomly divided into 8 different groups (n = 6) (Table 1) in a single-blind pattern by adopting a random distribution scheme with an equal number of male (n = 3) and female (n = 3) mice in each group (Miller et al. 2017): (i) Vehicle control group was administered 0.2% DMSO (i.p.) from day 1 to 14 and normal saline (s.c.) from day 1 to 7; (ii) LCD(3) group was administered lacidipine (3 mg/kg) from day 1 to 14 to observe the per se effects of the lacidipine on behavioral symptoms; (iii) Nic group served as nicotine-withdrawal control group that received (−)-nicotine hydrogen tartrate (3.35 mg/kg, t.i.d.) from day 1 to 7 only and drug vehicle from day 1 to 14; (iv) LCD(0.3)+Nic group was administered lacidipine (0.3 mg/kg) from day 1 to 14 and Nic (3.35 mg/kg, t.i.d.) from day 1 to 7; (v) LCD(1)+Nic group was administered lacidipine (1 mg/kg) from day 1 to 14 and Nic (3.35 mg/kg, t.i.d.) from day 1 to 7; (vi) LCD(3)+Nic group was administered lacidipine (3 mg/kg) from day 1 to 14 and Nic (3.35 mg/kg, t.i.d.) from day 1 to 7; (vii) Sertraline+Nic group was administered sertraline (40 mg/kg) from day 1 to 14 and Nic (3.35 mg/kg, t.i.d.) from day 1 to 7; (viii) Bay-K8644+LCD(3)+Nic group was administered Bay-K8644 (0.5 mg/kg) on day 13 and 14 before behavioral tests, lacidipine (3 mg/kg, 14 days) and (−)-nicotine hydrogen tartrate (3.35 mg/kg, t.i.d., 7 days).
Nicotine-withdrawal symptoms were induced in mice using protocol given by Aggarwal and Bansal (2018). Mice were exposed to (−)-nicotine hydrogen tartrate (3.35 mg/kg, s.c., t.i.d.) for seven days to develop nicotine dependence and thereafter, mice were refrained from further nicotine exposure to trigger symptoms of nicotine-withdrawal (Biala et al. 2014; Matta et al. 2007). After 48 h (i.e., day 9) of the last dose of (−)-nicotine hydrogen tartrate, animals in groups (i) – (vii) were observed for 30 min to assign withdrawal signs scores (somatic withdrawal signs) i.e., head shake, leg licking, rearing, grooming, jumping, and genital licking in a clean home cage (Isola et al. 1999; Biala et al. 2014). Each sign was assigned scores of one. Behavioral tests were conducted 60 min after drug treatments to assess the affective symptoms viz. memory, anxiety-like, and depressive symptoms in mice. All the groups were subjected to elevated plus maze (EPM) test, mirror chamber test, and tail suspension test (TST) on days 13 and 14. After behavioral tests, mice were euthanized by using the cervical dislocation method, and biochemical parameters of oxido-nitrosative stress were determined in the whole brain homogenate (Table 1).
Table 1. Experimental protocol to assess effects of lacidipine (LCD, Ca2+ antagonist) on nicotine-withdrawal signs and symptoms noted in mice that were given continuous exposure of (−)-nicotine hydrogen tartrate (Nic) at dose 3.35 mg/kg (s.c., t.i.d.) for 7 days.
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Day 1
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2
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3
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4
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5
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6
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7
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8
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9
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10
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11
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12
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13
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14
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Group (n = 6)
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(i) Vehicle control
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0.2% DMSO + Normal saline
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0.2% DMSO
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(ii) LCD(3)
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LCD (3 mg/kg)
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(iii) Nic
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Nic + DMSO
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0.2% DMSO
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(iv) LCD(0.3) + Nic
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LCD (0.3 mg//kg) + Nic
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LCD (0.3 mg//kg)
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(v) LCD(1) + Nic
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LCD (1 mg//kg) + Nic
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LCD (1 mg//kg)
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(vi) LCD(3) + Nic
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LCD (3 mg//kg) + Nic
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LCD (3 mg//kg)
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(vii) Sertraline + Nic
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Sertraline (40 mg/kg) + Nic
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Sertraline (40 mg/kg)
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(viii) Bay-K8644 + LCD(3) + Nic
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LCD (3 mg//kg) + Nic
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LCD (3 mg//kg)
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LCD (3 mg//kg) + Bay-K8644 (0.5 mg/kg)
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Vehicle control group was given drug vehicles and LCD(3) was administered LCD (3 mg/kg) for 14 days. Nic group was continuously exposed to nicotine for 7 days and thereafter, Nic administration was discontinued to trigger somatic and affective withdrawal signs. LCD (0.3, 1 and 3 mg/kg, i.p.) or sertraline (40 mg/kg, i.p.) were given for 14 days alongside Nic for 7 days in separate groups (iv – vii). In groups (i) – (vii), all the drugs were administered from day 1 onwards and somatic withdrawal signs were assessed on day 9. In group (viii), L-type Ca2+ agonist, Bay-K8644 (0.5 mg/kg), was injected before behavioral studies in mice that received LCD (3 mg/kg, 14 days) and Nic (dose 3.35 mg/kg, t.i.d. for 7 days) from day 1. All the groups were subjected to elevated plus maze (EPM) test on day 13 and 14, mirror chamber test on day 13, and tail suspension test (TST) on day 14.
Elevated plus maze test
Elevated plus maze test is widely used to assess spatial learning and memory in rodents across the globe. The standard protocol of EPM test was followed in the present study to assess memory abilities in mice (Bansal and Parle 2011). The EPM apparatus (wooden) consisted of two exposed sections (16 × 5 cm) and two enclosed sections (16 × 5 × 12 cm) contrary to each other hooked via an intermedial dais (5 × 5 cm), positioned at 40 cm height. Individual animals were placed at the edge of an exposed section facing opposite from the intermedial dais. Time (s) required by mouse to relocate from the exposed section into either of the enclosed sections with all its 4 paws was noted as transfer latency (TL). Mice not entering into either of the enclosed section within 90 s were gently guided into nearest enclosed section, and 90 s of TL was allotted. Each mouse was kept in the maze for an additional 10 s for exploration and thereafter transferred to their respective cages. During the acquisition trial (day 13) TL was observed (training session) for each moue. After 24 h of the acquisition trial, mice were evaluated for retention of this learned task (memory), and TL was noted (day 14). A significant decline in TL value during the retrieval trials indicated improvement in spatial memory (Itoh et al. 1990).
Mirror chamber test
For evaluation of anxiety-like symptoms, an approach-avoidance behavior of mice in response to the mirror was utilized by employing a mirror chamber apparatus. Its design consisted of an outer large compartment made of plywood (40 cm length × 40 cm breadth × 30.5 cm height) and an inner mirror-compartment (35 cm length × 35 cm breadth × 30 cm height) positioned at the center of the large compartment that allows a 5 cm passage between walls of both compartments. Another mirror (6th) was secured on the outer compartment wall in front of the exposed portion of the inner mirror-compartment. Individual mouse was positioned softly at a place in the passage at backside of mirror-compartment. Animal was permitted free access to all the parts if the apparatus including mirror-compartment for 5 min. Subsequently, (a) latency to enter the mirror chamber (s), (b) the number of entries, and (c) total time spent (s) in the mirror chamber were manually recorded (Toubas et al. 1990).
Tail suspension test
The method given by Steru et al. (1985) was implemented to measure passive motionless period of each animal that correlates to depression-like signs. Mice subjected to the short duration of inescapable stress show immobility which is a reliable index of depressive states. Individual animal was fastened (adhesive tape ~1 cm) by tip of the tail at an elevation of 58 cm above the table-top. Duration of static or motionless dangling of animal was measured in s for a 6 min period. A decrease in static period denotes antidepressant effect. The results of TST were stated as the duration of immobility (s).
Preparation of whole-brain homogenate
The mice were euthanized (n = 6) by cervical dislocation method. Instantly complete brain was harvested and bathed with freezing sterile isotonic normal saline and weight (g) noted. The brain was homogenized using tissue homogenizer (Remi Motors, Remi Electrotechnik, Vasai, India) in ice-cold 50 mM sodium-phosphate phosphate buffer (pH 7.40) at temperature 4°C to prepare 10% w/v brain homogenate. Subsequently, a clear supernatant was obtained by centrifuging (CPR-30 Remi Compufuge, Vasai, India) whole brain homogenate for 15 min at 4℃ at 12,000 × g force. The clear supernatant was separated for evaluation of biochemical parameters.
Estimation of lipid peroxidation in the brain
Thiobarbituric acid reactive substances (TBARS) reflect lipid peroxidation and relates with malondialdehyde (MDA) production (Ohkawa et al. 1979). The analyze mixture (final volume 4 ml) comprised 0.10 ml test sample (homogenate), 1.50 ml thiobarbituric acid (TBA, 0.8%), 1.50 ml glacial acetic acid (20%, pH 3.50), 0.20 ml sodium dodecyl sulphate (SDS, 8.10%), and 0.70 ml purified water. The analyze mixture in test-tubes was mixed vigorously, boiled on a water bath for 1 h (temperature 95°C), and cooled under flowing tap water. n-butanol and pyridine (15:1) solution was added (5 ml) and centrifuged for 10 min at 4,000 × g force. The optical density (O.D.) of MDA-TBA2 adducts (pink color) in the superior (n-butanol phase) 2 ml organic layer was noted at λmax = 532 nm by means of a double beam UV-spectrophotometer (Shimadzu UV-1700, Pharmaspec). TBARS (nanomole/mg protein) quantified using ε = 1.56×105 /M/cm.
Estimation of reduced glutathione (GSH) content
Test samples containing 1.0 ml of supernatant were precipitated using equivalent amount of 4% sulphosalicylic acid. The analyze mixture was cold-digested (4°C temperature) for 1 h and then centrifuged (4°C temperature) for 15 min at 2000 × g force. Supernatant was collected (0.10 ml) and mixed with 2.70 ml Na+-K+ PO43- buffer (0.31 M, pH 8.1) and 0.20 ml 5,5′-dithiobis(2-nitrobenzoic acid) (DTNB: 0.11 mM, pH 8.1). Standard curve of reduced GSH prepared (concentrations of GSH 0.2, 0.4, 0.6, 0.8, and 1 mM). The O.D. standard and tests were noted spectrophotometrically at λmax = 412 nm and the amount of reduced GSH (micromole GSH/mg protein) quantified (Ellman 1959).
Determination of superoxide dismutase (SOD) activity
The activity of superoxide dismutase (SOD) was estimated by following the method of Winterbourn et al. (1975). Briefly, the test-tubes (final volume 3 ml) containing 0.05 ml supernatant of homogenized whole brain, 0.05 ml riboflavin (0.12 mM), 0.10 ml nitroblue tetrazolium (NBT, 1.50 mM), 0.20 ml of 0.10 M ethylenediaminetetraacetic acid (EDTA, 0.30 mM sodium cyanide NaCN), and Na+/K+ PO43- buffer (67.0 mM, pH 7.81) q.s. were illuminated for 13 ± 3 min underneath 100 W fluorescent-tube (Bajaj®) and optical density variability (λmax = 560 nm) noted. The SOD in the sample impedes the reduction of NBT by O2ˉ and formazan synthesis. SOD activity (µM NBT reduced per min per mg protein) quantified using ε (formazan) = 15,000 /M/cm.
Determination of catalase (CAT) activity
The analyze mixture (3.0 ml) contained 0.05 ml test sample (supernatant), 1.10 ml hydrogen peroxide (H2O2 0.02 M) in Na+/K+ PO43- buffer (pH 7.80, 0.05 M), and 1.85 ml of 0.05 M Na+/K+ PO43- buffer (pH 7.0). Change in O.D. noted spectrophotometrically at λmax = 240 nm for 3 min at 30 s intermissions. Catalase (micromole H2O2 decomposed/min/mg protein) activity was quantified by means of ε = 43.60 /M/cm (Claiborne 1985).
Estimation of total nitrite content in the brain
The method given by Sastry et al. (2002) was adopted for determination of total nitrites. Briefly, the test-tubes containing 0.10 ml test samples (supernatant) or standard, alloy of Copper-Cadmium (150 mg), and 0.40 ml carbonic acid (H2CO3) buffer (pH 9.0) were subjected to incubation (1 h at ambient temperature). 0.10 ml NaOH (0.35 M) and 0.40 ml ZnSO4 solution (120.0 mM) were added and analyze mixture centrifuged at 4000 × g for 10 min and supernatant separated. Griess reagent (0.05 ml) was mixed with 0.1 ml of supernatant. After incubation of 30 min at room temperature in the dark, O.D. at λmax = 548 nm was determined spectrophotometrically. A standard curve of NaNO2 (concentration range 0.01-0.1 mM) was plotted, total nitrite content compared, and stated as μmole per mg of brain protein.
Estimation of total proteins in the brain of mice
The total protein content (mg/ml) was quantified using a typical curve of bovine serum albumin (BSA) with concentration range 0.2-2.4 mg/ml. The test blend was arranged using 0.25 ml test sample containing supernatant, Lowry’s reagent (5.0 ml), and Na+/K+ PO43- buffer (1.0 ml). After incubation (15 min, room temperature), Folin-Ciocalteu reagent (0.50 ml of 1.0 N) was added, mixed, and again incubated (30 min, room temperature). O.D. was determined spectrophotometrically at λmax = 650 nm (Lowry et al. 1951).
Statistical analysis
Data were analyzed by an experienced experimenter blinded to diverse treatments received by different groups of mice. The data were analyzed by one-way ANOVA followed by Tukey’s honest significant difference (HSD) post-hoc test or two-tailed paired t-test using software GraphPad Prism 5.0 (GraphPad Software Inc., California, USA). Two-way ANOVA and Bonferroni post-hoc test was used to evaluate the influence of sex on the outcome of different parameters studied in experiments. All results were expressed as mean ± standard error of mean (S.E.M.) and p < 0.05 was deemed to be statistically significant.