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50 values (obtained by GraphPad) for the displacement of the bound radioligand by increasing concentrations of the test compounds.
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Male Swiss albino mice (24-26 g) from Morini (San Polo d'Enza, Italy) were used. Ten mice were housed per cage. The cages were placed in the experimental room 24 h before the test for acclimatization. The animals were fed a standard laboratory diet and tap water ad libitum and kept at 22 ± 1 °C with a 12 h light/dark cycle, light at 7 a.m. All experiments were carried out in accordance with the NIH Guide for the Care and Use of Laboratory animals. All efforts were made to minimize animal suffering and to reduce the number of animals used. Mice were placed inside a stainless steel container, which was set thermostatically at 52.5 ± 0.1 °C in a precision water bath from KW Mechanical Workshop, Siena, Italy. Reaction times (s) were measured with a stopwatch before and 15, 30, 45, and 60 min after administration of analgesic drugs. The endpoint used was the licking of the fore or hind paws. Those mice scoring less than 12 and more than 18 s in the pretest were rejected (30%). An arbitrary cut-off time of 45 s was adopted to avoid injury. No sign of tissue injury was observed up to 45 s. Ten mice per group were tested.
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35648993945
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note
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The apparatus consisted of a base platform and a rotating rod with a diameter of 3 cm and a non-slippery surface. The rod was placed at a height of 15 cm from the base. The rod, 30 cm in length, was divided into five equal sections by six disks. Thus, up to five mice were tested simultaneously on the apparatus, with a rod-rotating speed of 16 rpm. The integrity of motor coordination was assessed on the basis of the number of falls from the rod in 30 s according to Vaught et al. Those mice scoring less than 3 and more than 6 falls in the pretest were rejected (20%). The performance time was measured before (pretest) and 15, 30, and 45 min after the beginning of the test. Ten mice per group were tested.
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