Experiment 3.2. Striatal ROS and MMP expression in rats chronically treated with doxycycline +L-DOPA
(i) MMP-2/MMP-9 in situ gelatinolytic zymography and ROS histochemistry was measured at the striatum slices of the experimental groups: 6-OHDA+chronic-vehicle+L-DOPA (n=7), 6-OHDA+chronic-doxycycline+L-DOPA (n=8) and the respective controls [no-lesion+chronic-vehicle+vehicle (n=6), 6-OHDA+chronic-vehicle+vehicle (n=6), 6-OHDA+chronic-doxycycline+vehicle (n=6)].
(ii) MMP-2 and MMP-9 substrate gel zymography was measured at the striatum tissue in the experimental groups: 6-OHDA+chronic-vehicle+L-DOPA (n=7), 6-OHDA+chronic-doxycycline+L-DOPA (n=6) and the respective controls [no-lesion+chronic-vehicle+vehicle (n=6), 6-OHDA+chronic-vehicle+vehicle (n=6), 6-OHDA+chronic-doxycycline+vehicle (n=6)].
Experiment 4 Impact of acute administration of COL-3 on LID expression once dyskinesia was present
In this experiment, a group of 6-OHDA+vehicle+L-DOPA (n=15) animals received L-DOPA for 14 days. To test the COL-3 effect (50 and 100 nmol - approximately 0.1 and 0.2 µg kg-1; Echelon Biosciences, Salt Lake, UT, USA, dissolved in saline), after L-DOPA treatment rats had an i.c.v. cannula (15-mm guide cannula, 22 gauge, C313/G/SPC; Plastics One Inc., Roanoke, VA) implanted above the left lateral ventricle (AP: + 0.4 mm, ML: −2.9 mm, DV: −4.0 mm relative to bregma (see the dopaminergic neuronal lesion with 6-OHDA section for details of the surgery). The cannula was stabilized with 5 micro screws and fixed with Jet Denture Repair Acrylic (Lang Dental, Wheeling, IL). The guide cannula was fitted with a 28-gauge inner stylet (Plastics One) to maintain cannula patency. The animals were allowed to recover for five to seven days.
Animals were administered COL-3 (50 and 100 nmol, n=7). The drug and the vehicle were injected into the ventricle of awake animals through a 15-mm-length stainless steel needle attached to an infusion pump 10 minutes before L-DOPA. To ensure that the investigator was blind to the rats’ individual treatment, COL-3 or vehicle was given randomly on either day 1 or day 2 of a 2 consecutive days testing period. On day 1, the animals received acute COL-3 (n=8), of either 50 or 100 nmol. The other animals (n=7) received vehicle before L-DOPA. A comparison between the AIMs scores among 6-OHDA+COL-3+L-DOPA vs. 6-OHDA+vehicle+L-DOPA was performed by one-way ANOVA-RM, followed by Bonferroni’s test.
The animals had their motor activity evaluated using the open field test, one hour after L-DOPA application.
Cannula placement was analyzed through striatal sections mounted on gelatinized slides using a light microscope (Leica Microsystems Launches Leica FW4000 - Cambridge, UK) following cresyl violet staining (Sigma-Aldrich). All the tested animals had the cannula inserted in the lateral ventricle, between AP−3.4 to −4.3 mm.