A whole-body exposure chamber containing the mice was mechanically ventilated (7025 rodent ventilators, Ugo Basile, Biological Research Instruments, Comerio, Italy) with either the smoke from five commercial Marlboro Red cigarettes (10 mg of tar, 10 mg of carbon monoxide and 0.9 mg nicotine) diluted 1:10 with air or air alone for 20 min. Mice were exposed to CS of five subsequent lit cigarettes for 20 min daily on three consecutive days in an acute or on each of five consecutive days per week for four months in a chronic approach.
Intratracheal (i.t.) administration of 80 µl PBS containing 10 µM 5-(3-Bromophenyl)-1,3-dihydro-2H-benzofuro-[3,2-e]-1,4-diazepin-2-one dissolved in DMSO (Tocris, Bristol, UK) or 80 µl PBS with 0.05% DMSO as vehicle control was performed 30 min prior to every smoke exposure. The amount of 10 µM chosen was based on preliminary dosing experiments (data not shown) according to results from an ATP-induced current inhibition assay applied with transfected HEK293 cells [19 (link)]. To minimize cage effects mice within the same cage were randomly allocated to the different treatment groups of the corresponding experiment.
Intratracheal (i.t.) administration of 80 µl PBS containing 10 µM 5-(3-Bromophenyl)-1,3-dihydro-2H-benzofuro-[3,2-e]-1,4-diazepin-2-one dissolved in DMSO (Tocris, Bristol, UK) or 80 µl PBS with 0.05% DMSO as vehicle control was performed 30 min prior to every smoke exposure. The amount of 10 µM chosen was based on preliminary dosing experiments (data not shown) according to results from an ATP-induced current inhibition assay applied with transfected HEK293 cells [19 (link)]. To minimize cage effects mice within the same cage were randomly allocated to the different treatment groups of the corresponding experiment.
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