Diazepam attenuates conditioned histamine release in guinea pigs

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Abstract

To clarify the possibility of pharmacological mediation on classical conditioning-associated asthmatic response, the effect of diazepam on an odor-induced conditioned histamine release was investigated in ovalbumin (OA)-sensitized guinea pigs, i.e. a model of bronchial asthma. The animals received conditioning sessions in which an antigen (OA) as the unconditioned stimulus and an odor (dimethylsulfide) as the conditioned stimulus (CS) were simultaneously inhaled. After the animals were intraperitoneally injected with saline or diazepam (2.5 or 5 mg/kg), they underwent exposure to the CS and blood collecting. This procedure was repeated three times in order that the animals would have each kind of injection. The animals injected with saline showed significantly higher levels of plasma histamine following the exposure to the CS as a conditioning effect compared with the baselines (P<0.05), whereas the group injected with diazepam (5 mg/kg) did not indicate such elevations. The suppressing effect of diazepam on the conditioned histamine release was also confirmed by a multiple regression analysis (5 mg/kg) and an analysis of covariance (2.5 and 5 mg/kg), even after adjustments for several factors regarding immunological sensitization and conditionability. The present study suggests that diazepam attenuates a conditioned histamine release.

Introduction

Classical or Pavlovian conditioning has gained increased importance in revealing an intimate relationship between the central nervous system and the immune system (Cohen et al., 1994). The phenomenology of conditioning of biological functions related to allergic reactions has also been characterized in recent years. Some investigators found that histamine or mast cell protease II was released following a classical conditioning procedure in guinea pigs (Russell et al., 1984) and rats (MacQueen et al., 1989). For example, Russell et al. (1984) reported that after a conditioning procedure in which nasal challenge of bovine serum albumin (BSA) was paired with the presentation of an odor, guinea pigs sensitized by intracutaneous BSA and Freund's adjuvant showed increased plasma histamine levels when presented with the odor alone. We also demonstrated that conditioned histamine release, in which an immunological challenge (ovalbumin, OA) as the unconditioned stimulus (US) was paired with the presentation of a neutral odor (dimethylsulfide, DMS, sulfur smelling) as the conditioned stimulus (CS), could occur in response to the CS after five CS–US pairings in the guinea pig, i.e. a model of bronchial asthma (Irie et al., 2001). In addition, we observed that the conditioned histamine release into the plasma, bronchoalveolar lavage fluid and lung tissue following the CS–US pairings was not reproduced under general urethane anesthesia (Irie et al., 2001). The findings suggest that the exacerbation of allergic reactions due to conditioning operation seems to depend on cognitive processes. If so, sedation which decreases cognitive processes may attenuate conditioned histamine release. In view of our previous experiment (Irie et al., 2001), the conditioned histamine release into plasma also may not be reproduced under sedation.

Diazepam, a commonly used sedative agent, has generally been considered to decrease memory and learning (Kleinknecht and Donaldson, 1975). Diazepam has been reported to show a dose-dependent impairment of cognition (Ghoneim et al., 1984). Moreover, diazepam (4 or 6 mg/kg) was found to impair olfactory cognitive processes in rats (Anglade et al., 1999). Taking these reports into consideration, it may be possible to consider that diazepam attenuates conditioned histamine release in a dose-dependent manner. Although numerous studies have been carried out on classical conditioning, very few reports investigated the effect of diazepam upon conditioned phenomena. Ervin and Cooper (1988) found that conditioned taste aversion of fluid consumption was blocked by pretreatment with diazepam (5 mg/kg) and other anxiolytic drugs in rats, whereas Gorczynski and Holmes (1989) reported that diazepam (2 μg/ml) did not show the blocking of antibody response to conditioned taste aversion in mice. Since the dose of diazepam, the investigated parameters and the species of animals were different between these studies, such factors may be associated with the different outcomes. Therefore, it seems to be of interest to investigate the effect of different doses of diazepam on conditioned histamine release.

To investigate this, however, it is required to consider the physiological effects of diazepam on airway response or histamine release. Benzodiazepines have been found to have several pharmacological actions, such as anxiolysis, sedation, muscle relaxation and ventilatory depression (Greenblatt and Shader, 1974), which include a possibility to worsen asthmatic responses. With respect to muscle relaxation relevant to ventilatory depression, Koga et al. (1992) reported that diazepam induced concentration-dependent relaxation of airway smooth muscle, and suggested that this effect was caused not via neural pathways or central and peripheral benzodiazepine receptors, but by a direct action on guinea pigs’ tracheal smooth muscle. Ventilatory depression has generally been considered to occur when high doses of benzodiazepines are administered intravenously; however, a low dose of oral diazepam (5 mg) was reported to inhibit ventilatory adaptation to mild hypoxia (Roggla et al., 1994). In addition, diazepam was reported to be unrelated to the changes in peak expiratory flow rate and airway resistance in asthmatics (Heinonen and Muittari, 1972). Thus, diazepam may not necessarily have an adverse effect on asthmatic responses. On the contrary, the agent may be useful to treat stress-related airway responses, since Portela et al. (2002) found that foot shock stress induced a significant increase in both edema and lymphomononucleated cells in airways in OA-sensitized rats, and diazepam suppressed such responses. In view of the findings of Portela et al. (2002), the possibility that diazepam itself may directly suppress histamine release from mast cells and basophils needs to be taken into account. However, diazepam has been reported to induce a small histamine release from human mast cells in vitro (Marone et al., 1993) and into plasma in vivo (Doenicke et al., 1993). It is therefore suggested that diazepam itself does not suppress histamine release into plasma at the anaphylactic phase.

The present study was designed to examine the effect of diazepam on the conditioned histamine release-evoking effects of a CS previously paired with an asthma-evoking US in guinea pigs. We investigated whether the conditioned histamine release into plasma could be reproduced or not under sedation by diazepam. Diazepam was chosen, because it has been well investigated in relation to airway function (Heinonen and Muittari, 1972, Koga et al., 1992, Roggla et al., 1994, Portela et al., 2002), histamine release (Doenicke et al., 1993, Marone et al., 1993), cognitive processes (Kleinknecht and Donaldson, 1975, Ghoneim et al., 1984, Anglade et al., 1999) and classical conditionings (Ervin and Cooper, 1988, Gorczynski and Holmes, 1989). With respect to the inhalation-induced allergic reaction, Chauveau et al. (1992) reported that bronchial provocation tests using aerosols showed marked differences among guinea pigs and also indicated large intraindividual changes in bronchial responsiveness. Taking into consideration these findings and the potential effect of the dose of diazepam on conditioned histamine release, we tried to measure the CS-related plasma histamine levels three times on different doses of diazepam-pretreatment according to a latin-square design in order to minimize possible interindividual and intraindividual variations of the OA-sensitization and plasma histamine levels.

Section snippets

General procedures

Twenty-four male Hartley guinea pigs (Seiwa Experimental Animal Institute; Fukuoka, Japan), 8 weeks old at the start of the experiments, were used. The animals were group-housed in standard wire laboratory cages. The environmental conditions were kept constant (light–dark cycle of 12:12, light on 07.00 h; temperature, 23±1 °C; humidity, 65±5%) throughout the experiments. Tap water and rodent chow were available ad libitum.

The guinea pigs were allowed more than 2 weeks to acclimatize to the

Results

In the first session, the levels of plasma histamine increased significantly from baseline following the exposure to the CS in the group injected with saline (t=−2.92, P<0.05), whereas the group injected with diazepam (5 mg/kg) did not indicate such elevations (Fig. 1). A significant difference in the plasma histamine levels after the inhalation of the CS was observed between the two groups in the first exposure to the CS (t=2.27, P<0.05); however, no differences were observed in either the

Discussion

In the present study, the effect of diazepam on classical conditioned histamine release was investigated in guinea pigs of the asthma model. As a result, it was suggested that diazepam, particularly a high dose of the agent (5 mg/kg), independently attenuated conditioned histamine release. The results are consistent with the findings of Ervin and Cooper (1988), who showed that conditioned taste aversion of fluid consumption was blocked by pretreatment with the same dose of diazepam in rats. A

Acknowledgements

We thank Prof. Masato Ikeda (Department of Occupational Health Economics, University of Occupational and Environmental Health) for his help in the statistical analysis.

References (21)

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