Impact of the acute administration of doxycycline on LID induced
ROS and MMPs once dyskinesia was present
ROS detectio n: The levels of red fluorescence of
dihydroethidium (DHE) oxidation as an indicator of ROS levels increased
in the lesioned striatum of parkinsonian animals treated with L-DOPA
(6-OHDA+vehicle+L-DOPA; Figs. 3-B, -G, arbitrary units). Acute
doxycycline treatment prior to L-DOPA led to a slight but significant
decrease in DHE oxidation (6-OHDA+doxycycline+L-DOPA; Figs. 3-C, -G; p
< 0.05). The increased level of ROS had no significant
correlation with dyskinesia severity (Fig. 3-H).
Detection of MMP-2/MMP-9 activity: A fluorescein isothiocyanate
(FITC) signal representing MMP-2/MMP-9 activity was observed in the
striatum of lesioned rats (6-OHDA+vehicle+vehicle; Figs. 3-D and -I) and
the striatum of rats expressing L-DOPA induced dyskinesia
(6-OHDA+vehicle+LDOPA; Figs. 3-E and -I). Doxycycline acute treatment
significantly reduced the MMP-2/-9 activity (Figs. 3-F and -I).
MMP-2/MMP-9 activity was highly correlated with dyskinesia severity
(r=0.9; p<0.01; Fig. 3-J).
MMP-2 and MMP-9 gel zymography: The presence of the MMP-2 (72
kDa) and the MMP-9 (97 kDa) was detected by substrate gel zymography in
the lesioned striatum (Supplementary Fig. 4). Striatal MMP-2 patterns
did not change wither following lesion (6-OHDA+vehicle+vehicle), or
L-DOPA treatment alone (6-OHDA+vehicle+L-DOPA) or with acute doxycycline
(6-OHDA+doxycycline+L-DOPA) (Supplementary Fig. 4-A). Contrastingly, the
striatal MMP-9 signal was increased after 6-OHDA-lesion but not modified
by L-DOPA treatment. Interestingly, acute doxycycline treatment
significantly reduced MMP-9 in lesioned rats but reached statistical
significance after L-DOPA treatment (p<0.05; Supplementary,
Fig. 4-B).
MMP-3 protein level: The western blot analysis revealed an
increased amount of MMP-3 in the lesioned-striatum of dyskinetic rats
(6-OHDA+vehicle+L-DOPA), which was significantly reduced by doxycycline
administration (6-OHDA+doxycycline+L-DOPA; p<0.0001; Fig.
3-K). The increase in MMP-3 levels was positively correlated with
dyskinesia severity (r=0.9; p<0.01, Fig. 3-L).