FIGURE LEGENDS
Figure 1. 6-OHDA nigrostriatal lesion characterization
and AIMs scores. Panel A shows the distribution of TH fibers
in the striatum (CPu) of controls (No-lesion- A and the inset)
shows intense TH-immunoreactive fiber terminals. Panel B shows
the effect of 6-OHDA on TH-immunoreactive fiber terminals. Likewise in
the SNc, TH-immunoreactive neurons were greatly reduced (D )
compared to controls (C ). Panels E and Frepresent the effects of doxycycline on the development of abnormal
involuntary movements (AIMs - E ) and time course of AIMs during
180 min (F ) induced by chronic administration of L-DOPA to
6-OHDA-lesioned rats. *P <0.001, Mann-Whitney, comparing
Vehicle+ L-DOPA (dyskinetic) with doxycycline-L-DOPA treated rats.
Data are mean ± SEM. DOXY: doxycycline; L-DOPA, L-3,
4-dihydroxyphenylalanine; 6-OHDA, 6-hydroxydopamine; VEH: vehicle; ALO
AIMs: global AIMs score including all AIM categories (axial, forelimb,
and orofacial movements).
Figure 2. NG2 distribution in striatum. A:Experimental design line. The striata was divided in dorsomedial (DM),
dorsolateral (DL), ventromedial (VM) and ventrolateral (VL) for
quantification analysis. B-E : Representative sections showing
NG2-glia immunostaining in the DL striatum from rats without lesion
(B- No lesion); lesioned with 6-OHDA toxin (C- 6OHDA);
lesioned with 6-OHDA+Vehicle+ L-DOPA treatment (D- 6OHDA+Vehicle
+L-DOPA); dyskinetic rats treated with doxycycline (E-6OHDA+L-DOPA+DOXY). Bar represents 20µm. F-I : Quantification of
NG2-glia by optic density (One-Way ANOVA) in the DM, DL, VM, VL striatum
of No lesion, 6OHDA, 6OHDA+DOXY, 6OHDA+Vehicle+L-DOPA and
6OHDA+DOXY+L-DOPA experimental groups. J-M : Spearman’s rank
correlation coefficient of NG2-glia with global AIMs scores axial,
forelimb, and orofacial dyskinesia scores in the DM, DL, VM, VL parts of
striatum. N : Relative NG2-glia protein expression level
(One-Way ANOVA) in DL striatum measured by Western blot (NG2: 26kDa; β
actin: 54kDa) in No lesion, 6OHDA, 6OHDA+DOXY, 6OHDA+Vehicle+L-DOPA and
6OHDA+DOXY+L-DOPA experimental groups of rats. *P <0.05,
6OHDA+LDOPA vs. all other groups. **P<0.001, 6OHDA+DOXY+L-DOPA
vs. 6OHDA+Vehicle+LOPA. #P<0.05, vs. No lesion. Data are mean
± SEM. DOXY: doxycycline; L-DOPA, L-3, 4-dihydroxyphenylalanine; 6-OHDA,
6-hydroxydopamine. DL= dorsolateral, DM= dorsomedial, VM= ventromedial;
VL= ventrolateral.
Figure 3. NG2-glia undergoes morphological changes in
the striata of rats presenting AIMs. A: Ramification analysis
figures; A´: Representative image of a NG2-glia cell (green)
used for the morphological analysis. Bar represents 5µm. A´´:Illustration of skeletonized cell; used for analysis to get information
of the cell morphology complexity. A´´´: Illustration of Sholl
analysis used to quantify process branching. For quantification, the
ramification index (RI) was calculated using the following formula: RI =
convex area/cell area (Heppner et al., 1998), where convex area is the
area of a polygonal object that is defined by the cells’ most prominent
projections and cell area is the area from soma. B-E: Data from
the ramification index of rats with L-DOPA-induced dyskinesia
(6OHDA+Vehicle+L-DOPA) and treated with Doxycycline (6OHDA+DOXY+L-DOPA)
in dorsomedial (DM), dorsolateral (DL), ventromedial (VM) and
ventrolateral (VL) striata areas. F: Morphometry analysis data
summary for DM, DL, VM and VL striatal areas including number of
branches, number of junctions, average branch length and average soma
area analyzed. *P<0.001 (One-Way ANOVA), vs.
6OHDA+Vehicle+LOPA. #P<0.001, vs. 6OHDA.
Data are mean ± SEM. DOXY: doxycycline; L-DOPA, L-3,
4-dihydroxyphenylalanine; 6-OHDA, 6-hydroxydopamine. DL= dorsolateral,
DM= dorsomedial, VM= ventromedial; VL= ventrolateral.
Figure 4. NG2-glia and GFAP double labeling in the
striatum. A-D : Graphs show the percentage of NG2-glia
co-labeling with GFAP in the total of NG2-glia in dorsomedial (DM),
dorsolateral (DL), ventromedial (VM) and ventrolateral (VL) striatum.E-I : Representative sections showing NG2-glia (green), GFAP
(red) staining and a merged image in DL striatum from No lesion, 6OHDA,
6OHDA+DOXY, 6OHDA+Vehicle+L-DOPA and 6OHDA+DOXY+L-DOPA experimental
groups of rats. Bar represents 20µm. Data are mean ± SEM. DOXY:
doxycycline; L-DOPA, L-3, 4-dihydroxyphenylalanine; 6-OHDA,
6-hydroxydopamine.
Figure 5. NG2-glia and OX-42 double labeling in the
striatum. A-D : Graphs show the percentage of NG2-glia
co-labeling with OX-42 in the total of NG2-glia in dorsomedial (DM),
dorsolateral (DL), ventromedial (VM) and ventrolateral (VL) striatum.E-I : Representative sections showing NG2-glia (green), OX-42
(red) staining and a merged image in DL striatum from No lesion, 6OHDA,
6OHDA+DOXY, 6OHDA+Vehicle+L-DOPA and 6OHDA+DOXY+ L-DOPA groups of rats.
Bar represents 20µm. Data are mean ± SEM. DOXY: doxycycline; L-DOPA,
L-3, 4-dihydroxyphenylalanine; 6-OHDA, 6-hydroxydopamine.