After the application of 6-OHDA, animals were treated with the following compounds: (i) VCE-003.2 (provided by Emerald Health Pharmaceuticals, San Diego, CA, USA) given orally at a dose of 20 mg/kg according to previous studies [29 (link)], initiating the treatment at 24 h after the lesion and daily repeating during 14 days; (ii) L-DOPA (Sigma-Aldrich Chem., Madrid, Spain) and benserazide (Sigma-Aldrich Chem., Madrid, Spain), given i.p. at the dose of 2 mg/kg in both cases according to previous studies [33 (link)], initiating the treatment 7 days after the lesion and daily repeating during 7 additional days (as indicated before, the use of a shorter treatment was necessary to diminish the occurrence of dyskinesia [33 (link)]); or (iii) the vehicle for VCE-003.2 (sesame oil) given orally during 14 days (50% of mice in this group) and the vehicle for L-DOPA/benserazide (0.9% saline) given i.p. during 7 days (remaining 50% of animals). At the end of the treatment (24 h after the last injection), mice were analysed in different behavioural tests just before being killed by rapid and careful decapitation. Their brains were rapidly removed and fixed for one day at 4 °C in fresh 4% paraformaldehyde (Sigma-Aldrich, Madrid, Spain) prepared in 0.1 M PBS, pH 7.4. Samples were cryoprotected by immersion in a 30% sucrose (Sigma-Aldrich, Madrid, Spain) solution for 48 h, and finally stored at −80 °C for immunohistochemical analysis in the substantia nigra.
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