Experimental Parkinsonism and dyskinesia
Dopaminergic neuronal lesion with 6-OHDA: Rats were rendered hemiparkinsonian as previously described (Gomes and Del Bel, 2003; Gomes et al., 2008; Padovan-Neto et al., 2015; Padovan-Neto et al., 2009;). The animals (n=270) were anesthetized with 2,2,2-tribromoethanol (Sigma-Aldrich, St. Louis, MO, USA‎) (250 mg kg-1, i.p.) and fixed into a stereotaxic frame (David Kopf, model USA, 9:57) with the incisor bar set at 3.3 mm below the interaural line. The rats received one deposit (coordinates from bregma in mm: AP = -4.3; LL = -1.6; DV = -8.3) of 2.0 μl 6-OHDA (Sigma-Aldrich, St Louis, MO, USA) into the left medial forebrain bundle as described by Gomes et al. (2008) (6-OHDA - 2.5 µg µl-1 in 0.9% NaCl supplemented with 0.02% ascorbic acid, 1 μL min-1). Following surgery, all rats were placed in clean cages on a warming pad for recovery (60 min), after which they were returned to group housing. A non-lesioned control group (n=24) was also included.
Two weeks later, the extent of the 6-OHDA-induced injury to the dopaminergic neurons was estimated using an apomorphine-induced rotation test (0.5 mg kg-1 in 0.9% NaCl, subcutaneous [s.c.], Sigma). Motor asymmetry was assessed using an automated system (Columbus Instruments International, Ohio, USA; Ungerstedt and Arbuthnott, 1970). Only those rats showing >90 total full contralateral turns over 45 min were selected for the study. Rats meeting this criterion have been reported to have a greater than 95% depletion of striatal dopamine (Chang et al., 1999; Kirik et al., 1998; Robinson and Becker 1983).
The lesion was confirmed histologically at the end of the behavioral tests by tyrosine hydroxylase (TH) immunohistochemistry. This confirmed that all rats in this study had more than 90% of diminution in TH immunoreactivity both in the dopamine depleted striatum and in the SNc.
Drug treatment The dosage regimen and routes of administration were based on previously published studies (Cenci et al., 1998; LazzarinI et al., 2013; Padovan et al., 2009; Worlitzer et al., 2013).
The preparation of L-DOPA used for animals was given orally as it is currently used for treating patients with PD. The concentration (20 mg kg−1) is similar to the usual regimen of L-DOPA in patients ranging from 0.2 to 1.6 g/day, which corresponds to about 1–20 mg kg-1 in animal models (Charvin et al., 2018; Lundblad et al., 2004). However, L-DOPA has low bioavailability when administered orally in comparison with the intraperitoneal route (Bredberg et al., 1994; Kurlan et al., 1988) and the oral route exhibits considerable variation in both the rate and extent of absorption. If oral L-DOPA was used at a lower concentration, the range of dyskinesia severity might vary greatly within a group of 6-OHDA-lesioned animals, which would compromise the evaluation of LID.
The daily dose of L‐DOPA (Prolopa dispersive, Hoffman-LaRoche, Brazil) was 20 mg kg-1 plus a peripheral decarboxylase inhibitor, benserazide (5 mg kg-1) (orally by gavage, dissolved in water). Doxycycline (40 mg kg-1, intraperitoneal [i.p.], Sigma) and COL-3 (i.c.v.) were dissolved in saline, since to ensure high lipid solubility with good penetration of the blood-brain barrier (Andersson and Alestig, 1976; Barza et al., 1975; Klein and Cunha, 1995; Liu et al., 2001).
L-DOPA-primed rats due to chronic L-DOPA treatment (14 days) were administered doxycycline or COL-3 after dyskinesia was present. The groups of parkinsonian rats submitted to doxycycline+L-DOPA (14 days, chronic) had not received treatment with L-DOPA previously. Doxycycline (i.p.) was administered 30 min before the L-DOPA or vehicle (gavage) in all experiments. Acute COL-3 (i.c.v.) was administered 10 min before the L-DOPA or vehicle.