MATERIALS AND METHODS

Subjects: Male Wistar rats weighing approximately 200 g, supplied from Animal Laboratory of School of Pharmacy Institute of Technology Bandung, were used. All animals were experimentally naive. Procedures for animal handling and treatment have been evaluated by the medical ethical committee of Hasan Sadikin General Hospital of Bandung. Experiments were conducted between August 2010 and April 2011.

Drugs: Nicotine [(-)-nicotine di(+)tartrate salt] was purchased from Sigma Chemical. Sodium diclofenac was a generous gift from Dexa Medica (Jakarta, Indonesia). The drugs were dissolved in physiological saline and injected intraperitoneally (i.p.) at a volume of 1 mL kg^-1 body weight. VCO, obtained from School of Life Sciences and Technology, Institute of Technology Bandung, was given orally at a dose of 5 mL kg^-1.

Apparatus: The place conditioning apparatus consisted of three distinct compartments (separated by guillotine doors). The overall dimensions of the apparatus were 30x30x70 cm (HxWxL). The center compartment of 10x10 cm dimension was gray with a smooth PVC floor. The choice compartments were 30 cm long. One compartment was all black with a stainless steel grid rod floor consisting of 4.8 mm rods, placed 16 mm on center, while the other was all white with a 0.25x1.25 cm stainless steel mesh floor. The apparatus had clear acrylic lid for animal loading.

Place conditioning training: On day, 1 rat were injected with either nicotine (0.5 mg kg^-1) or saline and immediately confined to one of the choice compartment for 30 min. The specific drug-paired chamber for each animal was randomly assigned, to give unbiased nature of this CPP design. Four hours later, the rats were injected with the alternate condition: rats receiving drug in the morning session received saline in the afternoon session and viceversa Rate then immediately confined to the opposite choice compartment for 30 min. This procedure was repeated for the next four days (day 2-5) so that each rat received a total of five drug and five saline sessions. The sequence of drug injections differed across and within rats on these conditioning days to eliminate any potential order effects. The treatment condition was altered each day so the rats did not receive the same treatment condition at the same time each day.

Preference test: On day, 6 rats were tested for post conditioning side preference. Following a 5 min acclimation period in the center gray compartment, the guillotine doors were lifted and rats were allowed to access to all three compartments for 15 min. The times spent in both compartments were recorded. Preference scores, the index of rewarding property in CPP paradigm, were then determined by dividing sojourn time in the drug-paired compartment to the total time spent in both drug-paired and saline-paired compartments during test session, using the following simple formula:

VCO (5 mL kg^-1) was administered orally to rats in test group 30 min prior to each nicotine injection during conditioning training. In different test group, instead of VCO, the reference drug diclofenac at 3.2 mg kg^-1 was given intraperitoneally.

Relapse test: Following conditioning test, the rats in this batch entered a 7-day drug free period. On the following day, they were challenged with nicotine at 0.5 mg kg^-1 intraperitoneally. VCO (5 mL kg^-1) was administered orally to rats in test group 30 minutes prior to nicotine challenge. In different test group, the reference drug diclofenac at 3.2 mg kg^-1 was given intraperitoneally.

Statistical analysis: Data were expressed as preference score, which was calculated dividing sojourn time (sec) in drug-paired compartment by total sojourn time(sec) in both drug-paired and saline-paired compartments. One-way Analysis of Variance (ANOVA) was used to compare means of preference score and Fisher’s PLSD was used for post hoc test (Anggadiredja et al., 2003), using Statview statistical software.

RESULTS

As depicted in Fig. 1, following a 5-day period of conditioning training, the preference score of rats receiving repeated nicotine treatment increased significantly, reaching a value of 0.516+0.195, compared to the pre-conditioning training score of 0.332±0.097.

Fig. 1:	Effects of VCO on the of nicotine dependence in rats as tested with conditioned place preference (CPP) paradigm. VCO (5 mL kg-1, p.o.) or diclofenac (3.2 mg kg-1, i.p) was given 30 min before each nicotine (0.5 mg kg–1, i.p.) injection during the 5-day period of preference training. *, **p<0.05 and 0.01, respectively (one-way ANOVA followed by Fisher’s PLSD)

Fig. 2:	Effects of VCO on nicotine relapse in rats as tested with conditioned place preference (CPP) paradigm. VCO (5 mL kg-1, p.o.) or diclofenac (3.2 mg kg-1, i.p.) was given 30 min before nicotine (0.5 mg kg-1) challenge delivered after a 7-day period of nicotine abstinence. *, **, ***p<0.05, 0.01, and 0.001, respectively followed by Fisher’s PLSD)

Figure 1 further revealed significant effect of VCO as well as diclofenac treatments, which decreased the preference score to 0.233±0.138 and 0.243±0.066, respectively (F(3,26) = 5.509, p = 0.0046, one-way ANOVA, post hoc Fisher’s PLSD).

Figure 2 describes that when the rats were made nicotine-free during a 7-day period of abstinence, the preference score was significantly decreased to a preconditioning-training level of 0.291±0.175. The score was again increased significantly (to as high as 0.553±0.257) when the rats were challenge with nicotine on the following day after the abstinence period. Figure 2 further describes, treatment with VCO or diclofenac before nicotine challenge resulted in significant decrease in preference score to 0.317±0.084 and 0.227±0.099, respectively, (F(5,54) = 5.571, p = 0.0003, one-way ANOVA, post hoc Fisher’s PLSD).

DISCUSSION

Result which showed the increase in preference score in rats treated repeatedly with nicotine corroborates the rewarding effect of nicotine. The same observation has been reported in early study by Shoaib et al.( 1994) which showed that CPP was produced in subjects having nicotine history with a dose range of 0.4-0.8 mg kg^-1, within which the current experimental dose (0.5 mg kg^-1) was used. This dose has also been used in previous CPP study (Risinger and Oakes, 1995; Grabus et al., 2006). Larger dose of nicotine of as high as 2 mg kg^-1 used in CPP experiment, on the other hand, produced conditioned place aversion, instead of preference (Le Foll and Goldberg, 2005).

The rewarding characteristic of nicotine was further confirmed when the drug was re-introduced to the abstinent rats previously exposed to repeated nicotine. Such an observation has been reported in a number of earlier works by Biala and Budzynska (2006, 2008), Fattore et al. (2009), Biala et al. (2009) and Budzynska et al. (2009). In clinical setting, this phenomenon of relapse has been a main hurdle in the treatment of drug addiction. Thus, addicts who have been drug-free, even for years, may still resume the drug taking habit when they are exposed to the drug or drug cues.

The present results showed that pretreatment with VCO prevent the development of nicotine dependence, as shown by the decrease in preference score in rats receiving VCO prior to every nicotine dose. VCO also prevented the increase in preference score in abstinent rats challenged with nicotine injection, indicating a blocking effect of VCO on relapse to nicotine.

It was only recently demonstrated that VCO has analgesic, antipyretic as well as antiinflammatory effects (Intahphuak et al., 2010). This range of pharmacological activities are the characteristics of non-steroidal antiinflammatory drugs, whose effects are known to interfere with the arachidonic acid cascade. In the past several years, the involvement of this endogenous metabolic pathway in the phenomena of dependence and relapse of addictive substances has been shown. Thus, prostaglandin, an end product of the cyclooxygenase pathway of the arachidonic acid cascade, has been shown to prevent the expression of withdrawal signs in mice made dependent on the cannabinoid agent delta-9 tetrahydrocannabinol (THC, a psychoactive ingredient of marihuana). This was confirmed by the withdrawal-inducing activity of diclofenac in mice repeatedly treated with delta-9 THC (Anggadiredja et al., 2003). Using withdrawal expression as indicator, it was further found that the signs were expressed when a group of cannabinoid-naive mice were treated consecutively with diclofenac and a cannabinoid receptor antagonist (Anggadiredja et al., 2005). With regard to relapse, a previous work with methamphetamine further corroborate the essential role of the arachidonic acid cascade. Using self-administration paradigm, in which rats were trained to actively deliver methamphetamine, it was found that diclofenac prevented methamphetamine-seeking behavior following the challenge of a small dose of this addictive substance in the drug-abstinent rats (Anggadiredja et al., 2004a).

The aforementioned works were also the demonstration of cross talk between the arachidonic acid cascade and endocannabinoid system. This endogenous interaction has also been demonstrated by Yamaguchi et al. (2001, 2004). Recently, Merritt et al. (2008) have reported that endogenous cannabinoid modulated nicotine CPP, corroborating the essential of cannabinoid-prostanoid interaction in rewarding property of psychoactive drugs and nicotine in particular.

Diclofenac was used in present study as reference substance for VCO effect on the rewarding property of nicotine. This was based on previous results which demonstrated the effectiveness of diclofenac in ameliorating dependence and reinforcing properties of several psychoactive drugs. Judging from the present results which showed that the effect of VCO was comparable with that of diclofenac, one may hypothesize that VCO interfered with the arachidonic acid cascade machinery. One possible explanation might reside in the common lipid nature of VCO (Dayrit et al., 2008) and the arachidonic acid (Morrow and Roberts, 2001). Thus, it might be that lipid components of VCO served as the source for arachidonic acid synthesis, whose downstream processes might eventually led to silencing effect on the preference to nicotine-paired CPP compartment.

The approach applied in the present study is closely related with learning-memory aspect, because in the development of dependence the rats were let to learn to associate drug to certain cue, in this case the drug-paired compartment of CPP apparatus. Indeed, as indicated in the previous study by Anggadiredja et al. (2004b), it is the memory that perpetuate the engagement between drug and drug users. Matsumoto et al. (2004) has indicated the role the arachidonic acid cascade in memory formation. Taking into consideration the possibility of VCO to interfere with the arachidonic acid cascade, one may further hypothesize that it also plays a role in the learning and memory aspects of drug dependence.

Taken together, while still at the very early stage, results of the present study are in favor with the evidence reported so far on interaction among dependence, memory and the arachidonic acid cascade metabolic pathway. VCO, whose preventive effects on nicotine dependence and relapse was demonstrated in this study, might have an essential role in the modulation of the arachidonic acid cascade by acting as a source of fatty acid.

CONCLUSION

The present work demonstrated, for the first time, preventing effects of VCO on nicotine dependence as well as relapse, as studied using conditioned place preference paradigm in rat model. The results may further lay foundation for the development of agent that can help addicts quit smoking and for combating drug addiction in general.

ACKNOWLEDGMENT

Funding for this work was supported by research grant scheme from ITB Alumni Association.