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.