Legend of the figures
Figure 1. Doxycycline acute administration reduced LID without compromising the beneficial motor effects. 6-OHDA lesioned rats received a daily administration of L-DOPA (20 mg kg-1) for 14 days to establish the expression of L-DOPA induced dyskinesia.(A) Sum of the AIMs score or ALO AIMs (axial, forelimb, and orofacial) are presented. The score was measured on days 1, 7, 14 of L‐DOPA administration (n=11) over 180 min. Doxycycline was inoculated in the animal 30 min before L-DOPA (6-OHDA+doxycycline+L-DOPA). AIMs expression decreased by 89% (one-way ANOVA-RM p<0. 0001; data presented as the mean±SEM) compared to 6-OHDA+vehicle+L-DOPA. (B) Time course of the appearance of dyskinetic manifestation with 6-OHDA+doxycycline+L-DOPA compared to 6-OHDA+vehicle+L-DOPA (p<0.001) across time (p<0.05, two‐way ANOVA-RM followed by Bonferroni posthoc test). (C)L-DOPA administration significantly improved performance in the stepping test; doxycycline-maintained the beneficial L-DOPA motor effects (p<0.01; one-way ANOVA followed by Bonferroni test; data are presented as the mean±SEM). (D) Doxycycline does not alter the L-DOPA effect on the distance traveled (p<0.0001; one-way ANOVA followed by Bonferroni; data are presented as the mean±SEM) *compared to 6-OHDA+vehicle+L-DOPA; #compared to 6-OHDA+doxycycline+L-DOPA Δ- compared to no-lesion+vehicle+vehicle. AIMs: abnormal involuntary movements; DOX: doxycycline; L-DOPA, L-3, 4-dihydroxyphenylalanine; RM, repeated measures; 6-OHDA, 6-hydroxydopamine.
Figure 2. Reduction of LID markers in the striatum of rats expressing LID after the acute administration of doxycyclinePhotomicrographs of the medial dorsal striatum (coronal sections - 0.10 mm with respect to bregma) from rats sacrificed 1 h after the last L-DOPA injection. Doxycycline treatment prior to L-DOPA reduced immunoreactivity for: FosB (A, B) ; OX-42 (D, E) ; GFAP(G, H) and COX-2 (J, K) . The bar charts (C, F, I, L, respectively) show the quantification of FosB, OX-42, GFAP, and COX-2 immunopositive cells. 6-OHDA+vehicle+L-DOPA group is represented in (A, D, G, J) and 6-OHDA+doxycycline+L-DOPA in(B, E, H, K) . A two-way ANOVA followed by the Bonferroni test was used to check for statistical significance (p<0.05); n=7-10/group; *compared to 6-OHDA+vehicle+vehicle; #compared to 6-OHDA+vehicle+L-DOPA; OX-42, CD11b/c beta-integrin marker of microglia; GFAP, glial fibrillary acid protein; COX-2, cyclooxygenase-2. Inset bar: 10 μm.
Figure 3. Reduction of ROS and MMPs in the striatum of rats expressing LID after the acute administration of doxycycline. ROS, MMP-2/MMP-9, and MMP-3 levels increased in the striatum of rats with dyskinesia that was decreased by doxycycline. (A-F)Representative image from the dopamine-denervated striatum of L-DOPA-treated rats (-0.10 mm relative to bregma; Paxinos and Watson, 1997) without and with doxycycline treatment illustrating ROS (redA-C ) and MMP-2/MMP-9 gelatinolytic activity (green D - F ) expressed as arbitrary fluorescence units. The graphics show the quantification of ROS and MMP activity (G, I, K) and the correlation between them and the dyskinesia index (H, J, L) . The 6-OHDA+vehicle+vehicle group is represented in (A, D); 6-OHDA+vehicle+L-DOPA in (B, E) and 6-OHDA+doxycycline+L-DOPA in (C, F). A one-way ANOVA followed by the Bonferroni test was used to check for statistical significance (p<0.05). There was no correlation between the dyskinesia sum of the score of rats and ROS fluorescence intensity (p=0.3; r=0.3; H ). Pearson’s correlation coefficient was significant between the sum of the dyskinesia score and gelatinolytic activity (p<0.0001; r=0.9; J ). MMP-3 expression (p<0.0001, r=0.9, L ) was revealed. Each point represents an individual rat. *compared to 6-OHDA+vehicle+vehicle; #compared to 6-OHDA+vehicle+L-DOPA; Δcompared to no-lesion+vehicle+vehicle (n=6-7/group). AIMs, abnormal involuntary movements; DOX, doxycycline; L-DOPA, L-3,4-dihydroxyphenylalanine; MMP, matrix metalloproteinase; ROS, reactive oxygen species.
Figure 4. Doxycycline/L-DOPA chronic co-treatment of parkinsonian rats since the first day of receiving L-DOPA block LID development and the increase of ROS and MMPs in the striatum (A)Cumulative total score of the axial, limb and orofacial AIMs evaluated on days 1, 7, and 14 after L-DOPA treatment. Doxycycline co-treatment blocked the development of L-DOPA induced dyskinesia (p<0.0001, two-way ANOVA-RM, Bonferroni posthoc test). Data are presented as the mean±SEM. (E, F, G) Representative images from the dopamine-denervated striatum of rats (-0.10 mm relative to bregma) illustrating ROS detection (red C, D ) and MMP-2/-9 gelatinolytic activity (green F, H ) measured as arbitrary fluorescence units. The 6-OHDA+chronic-vehicle+vehicle group is shown in(C, F) ; 6-OHDA+chronic-vehicle+L-DOPA in (D, G) and 6-OHDA+chronic-doxycycline+L-DOPA in (E, H). The graphics show the quantification of ROS and MMP activity (I, K) and the correlation between them and the dyskinesia index (J, L) . A one-way ANOVA followed by the Bonferroni test was used to check for statistical significance (p<0.05). Pearson’s correlation coefficient was significant between AIMs intensity and ROS (p<0.0001; r=0.9) and AIMs intensity and gelatinolytic activity (p<0.0001, r=0.7). All molecular markers are expressed in the denervated striatal areas. *compared to 6-OHDA+chronic-vehicle+vehicle; #compared to 6-OHDA+chronic-vehicle+L-DOPA; Δcompared to no-lesion+chronic-vehicle+vehicle; +compared to 6-OHDA+chronic-vehicle+L-DOPA at 1 Day of treatment; &compared to 6-OHDA+chronic-vehicle+L-DOPA at 7 Days of treatment (n=6-7/group) AIMS, abnormal involuntary movements; L-DOPA, L-3,4-dihydroxyphenylalanine; MMP, matrix metalloproteinase; ROS, reactive oxygen species; chDOX, chronic doxycycline.
Figure 5. COL-3 acute administration reduces LID in animals expressing LID. After L-DOPA induced dyskinesia had been established, the animals received two different doses of COL3 (50 or 100 nmol approximately 0.1 and 0.2 µg Kg-1intracerebroventricular) before L-DOPA. (A) Sum of the AIMs score (axial, forelimb, and orofacial) are presented. COL-3 (100 nmol) treatment significantly reduced the sum of AIMs over the 180 min observation period (p<0. 01; paired t-test n=5; p<0.05 RM one-way ANOVA n=7) compared to the measurement the previous day. The points represent data from individual animals.(B) Time course of the appearance of dyskinesia with 6-OHDA+COL-3+L-DOPA compared to 6-OHDA+vehicle+L-DOPA across the time period (p<0.01, two‐way ANOVA-RM followed by Bonferroni posthoc test). AIMS were evaluated on days 1, 7, and 14. Data are expressed as the mean ±SEM; p<0.05 #compared to 6-OHDA+vehicle+L-DOPA. AIMs, axial, limb and orofacial abnormal involuntary movements; COL-3, CMT-3,6-demethyl-6-deoxy-4-de[dimethylamino]-tetracycline; L-DOPA, L-3,4-dihydroxyphenylalanine; VEH, vehicle.