A multidisciplinary in vivo
and ex vivo approach has been used to evaluate the general outcome of the treatment on disease-sensitive indices. The final aim was to evaluate the possible presence of a synergistic action between the two compounds that may justify their combined use in patients. All experiments were conducted in accordance with the Italian Guidelines Saracatinib research buy for the use of laboratory animals, which conform with the European Community Directive published in 1986 (86/609/EEC). Most of the experimental procedures used conform the standard operating procedures for preclinical test in mdx mice available on http://www.treat-nmd.eu/research/preclinical/SOPs/[2,32]. Animal groups, treadmill running and drug treatment Male mdx and wild type (WT, C57/BL10ScSn) mice of 4–5 weeks of age (Charles River, Italy for Jackson Laboratories, USA), homogeneous for body weight were assigned to ‘exercised’ and ‘sedentary’ groups. The groups of exercised mice underwent a 30 min running on an horizontal treadmill (Columbus Instruments, USA) at 12 m/min, twice a week, for 4–8 weeks [8,33] and were composed click here by seven vehicle-treated
and six prednisolone-taurine-treated mdx mice. Based on previous results , we chose the dose of 1 mg/kg i.p. for PDN, while taurine was administered orally in chow-enriched pellets at the maximal dose of 1 g/kg/day. Both compounds have been already tested singularly in exercised mdx mice . However, in order to avoid any bias due
to variability of experimental conditions, two additional groups of exercised mdx mice were used. One group was made of five animals treated only with 1 mg/kg PDN i.p. while the other group of four animals received only taurine-enriched MycoClean Mycoplasma Removal Kit food up to 1 g/kg/day. The treatment started 1 day before the beginning of the exercise protocol, and continued until the day of sacrifice. When necessary, age-matched untreated exercised WT mice were also used. ‘Sedentary’ mdx (vehicle-treated or not) and WT mice were left free to move in the cage, without additional exercise and monitored at the same time points of exercised counterparts, according to the experimental need. Every week all mice were monitored for body weight and fore limb force by means of a grip strength meter (Columbus Instruments, USA); the end of the 4th week was considered for statistical analysis [8,34]. At the end of the 4th week of exercise/treatment the ex vivo experiments were also started. The animals continued to be exercised/treated until the day of sacrifice and were used for the ex vivo experiment within the 8th week. Muscle preparations Animals of 8–12 weeks belonging to the different groups were anesthetized with 1.2 g/kg urethane i.p. Extensor digitorum longus (EDL) muscle of one hind limb was removed and rapidly placed in the recording chamber for the electrophysiological recordings.