It is widely accepted that atopic dermatitis is a skin disease of itching, that is, a sensation causing the urge to scratch. However, no useful method for the measurement of itching in animals has been reported. Kuraishi et al. demonstrated that a subcutaneous injection of compound 48/80 into the rostral back of the mouse produced scratching of the injected site by the hind paws, which is an itch-associated behavior (1). Recently, we found that inhibition of compound 48/80-induced scratching behavior is mainly due to histamine H1-receptor antagonistic activity and not to the sedative action of the drugs (2). The present study was undertaken to clarify the role of histamine in generating the scratching behavior induced by compound 48/80 in mice.
Materials and Methods
1. Compound 48/80-induced scratching behavior
The animals used were female BALB/c strain mice. Before the experiment, the animals were put into an observation cage for about 10 min for acclimatization. With the use of a 27 gauge needle, 0.05 ml of compound 48/80 was injected subcutaneously into the rostral part of the back. Immediately after injection, the animals were put into the observation cage and scratching behavior was measured for 60 min. Scratching behavior was observed in accordance with the method of Kuraishi et al. (1) i.e., scratching of the injected site with the hind paws was counted. Test drugs were suspended in 5% gum arabic and were administered orally 1 h before compound 48/80 injection.
2. Compound 48/80-induced vasal permeability of the skin
The animals used were female BALB/c strain mice. After subcutaneous injection of 0.05 ml of compound 48/80 into the rostral part of the back, a 1% saline solution of Evans blue was injected intravenously into each animal. The animals were killed 60 min later, and the diameters of the ÒBluingÓ reaction at the compound 48/80 injection site were measured.
3. Histamine content
Mice were killed by exsanguination under ether anesthesia. Dorsal skin was harvested quickly and rinsed with ice-cold 0.9% NaCl containing semicarbazide dihydrochloride for inhibition of histamine oxidase. Each sample was homogenized in 10 x volume of 0.8 N perchloric acid with a Polytron homogenizer operated at maximal speed in an ice bath. The homogenate was centrifuged at 1,500 x g for 10 min at 4ûC. An aliquot (1 ml) of the clear deprotenized supernatant was separated. Histamine in the supernatant was measured by an automated fluorometric assay (3).
Repeated injection of compound 48/80 resulted in 62% and 75% decrease in the number of scratching behavior on day 6 and day 12, respectively (Fig. 1). The same was observed with regard to the skin histamine content, i.e., the skin histamine content was significantly decreased by repeated injection of compound 48/80 (Fig. 1).
Diphenhydramine caused a dose-related inhibition of compound 48/80- induced scratching behavior and an increase of vasal permeability and at doses of 1 and 3 mg/kg, it significantly decreased these responses (Fig. 2). Chlorpheniramine inhibited compound 48/80-induced scratching and an increase of vasal permeability similarly to diphenhydramine (Fig. 2).
Azelastine, histamine H1-receptor antagonist having antiallergic activity (an inhibition of mast cell degranulation), also inhibited compound 48/80-induced scratching behavior and an increase of vasal permeability (Fig. 3). As shown in Fig. 3, terfenadine at doses of 1, 3 and 10 mg/kg significantly inhibited these responses.
Both epinastine and astemizole, at doses of 3, 10 and 30 mg/kg, significantly inhibited compound 48/80-induced scratching behavior and an increase of vasal permeability (Fig. 4).
The effect of tranilast was relatively weak (data not shown). To test whether a central depressant drug inhibits the scratching behavior induced by compound 48/80 or not, diazepam was given p.o. at 0.3 mg/kg it caused no significant inhibition of the scratching behavior. At a dose of 1 mg/kg p.o., however diazepam caused a significant inhibition of scratching, but showed significant muscle relaxant activity (data not shown).
Discussion and Conclusion
In the present study, we found that classical histamine H1 receptor antagonists, diphenhydramine and chlorpheniramine, inhibited compound 48/80-induced scratching behavior and an increase in vasal permeability. It is well known that these drugs show a sedative effect as well as a histamine H1-receptor antagonistic action. Therefore, it was thought that the inhibition of scratching behavior induced by compound 48/80 occurred via a histamine H1-receptor antagonistic action or sedative effect. However, as shown in the text, non-sedative histamine H1-receptor antagonists such as terfenadine, epinastine and astemizole also inhibited this response to the same extent as diphenhydramine and chlorpheniramine. In association with this, diazepam, a sedative with no histamine H1-receptor antagonistic activity, had no or little effect on experimental pruritus induced by histamine (4). Therefore, it may be concluded that inhibition of compound 48/80-induced scratching behavior cannot be ascribed to the sedative action of the drugs. Azelastine was also found to cause inhibition of compound 48/80-induced scratching behavior and an increase of vasal permeability. Azelastine (5) and non-sedative histamine H1-receptor antagonists, terfenadine (6), epinastine (7) and astemizole (8) that show both histamine H1-receptor antagonistic activity and antiallergic activity that defined the inhibition of chemical mediator release from mast cells or basophils, caused almost the same effect as diphenhydramine and chlorpheniramine. In addition, tranilast, antiallergic drugs without histamine H1-receptor antagonistic activity, also possessed a relatively weak inhibitory effect on these response. From these findings, it can be assumed that the contribution of an antiallergic effect was relatively small for compound 48/80-induced scratching behavior and an increase of vasal permeability. In conclusion, inhibition of compound 48/80-induced scratching behavior and an increase of vasal permeability occurred mainly via H1-antagonistic activity.
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|Sugimoto, Y.; Nakamura, Y.; Kamei, C.; (1998). Involvement of histamine on compound 48/80-induced skin reactions in mice. Presented at INABIS '98 - 5th Internet World Congress on Biomedical Sciences at McMaster University, Canada, Dec 7-16th. Available at URL http://www.mcmaster.ca/inabis98/|
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