Tissue collection and processing
Rats were anesthetized with tribromoethanol (1.5 g kg-1, Sigma-Aldrich,
St. Louis, MO, USA) 60 min after the last L-DOPA injection before being
sacrificed.
Tissue collection for immunohistochemistry: Anesthetized rats
were perfused transcardially with 100 mL of Kreb’s-ringer buffer and 200
mL of buffered picric acid-paraformaldehyde fixative room temperature
(Somogyi and Takagi, 1982: one liter of fixative was made up by mixing
in the following order: 500 ml of 0.2 M sodium phosphate buffer (pH
7.4), 150 ml of saturated picric acid in distilled water; 348 ml of
paraformaldehyde solution containing 40 g of depolymerized
paraformaldehyde and 2 ml of 25% glutaraldehyde). The final pH was
between 7.2 and 7.4, and the final concentrations were 4.0%
paraformaldehyde, 0.05% glutaraldehyde, and 0.2% picric acid.
After perfusion, the brain was removed from the skull and fixed by
immersion in the same fixative for 60 min at 4 oC.
Then, it was cryoprotected in a 30% sucrose solution in 0.1 M phosphate
buffer for 24 h. The brains were quickly frozen in isopentane cooled in
liquid nitrogen (−40 °C, Sigma-Aldrich, St. Louis, MO, USA) and stored
at −80 °C until histological processing. Serial coronal sections (25 µm)
were cut using a freezing microtome (Leica, model CM1850) throughout the
rostrocaudal extent of the striatum and the SNc (Paxinos & Watson,
2004). The sections were collected in an anti-freeze solution (50%
phosphate buffer, 0.05 M pH 7.3, 30% ethylene glycol, 20% glycerol)
for preservation and subsequent processing.
Immunohistochemical reaction: Immunostaining was performed on
free-floating sections with standard avidin-biotin protocols (Bortolanza
et al., 2015 a, b; Gomes et al. 2008) with the antisera presented in
Supplementary Table 1.
Free-floating sections were submitted to antigen recovery by heating for
30 min in a water-bath at 60 ◦C in 0.1 M citrate
buffer with a pH of 6.0. After rinsing in the washing buffer
(phosphate-buffered saline 0.1 M (PBS – sodium chloride NaCl, (mw: 58.4
g/mol); potassium chloride KCl (mw: 74.551 g/mol); sodium hydrogen
phosphate Na2HPO4 (mw: 141.96 g/mol); monopotassium phosphate KH2PO4
(mw: 136.086 g/mol)) + 0.15% Triton-X100; pH 7.4), the sections were
incubated for 30 min with 1% hydrogen peroxide (diluted in washing
buffer) to block endogenous peroxidase activity. Nonspecific binding
sites were blocked by incubation in a solution containing 2% bovine
serum albumin plus 5% normal serum (from the species of origin of the
secondary antibody) in washing buffer for 60 min. Next, the sections
were incubated with primary antibody against tyrosine hydroxylase (TH),
FosB, glial fibrillary acidic protein (GFAP to reveal astrocytes), OX-42
(CD11b/c equivalent protein of microglia to reveal microglia) or
cyclooxygenase-2 enzyme-labeled cells (COX-2). After incubation in the
primary antibody for 24 h, the sections were successively rinsed in the
washing buffer and incubated in biotinylated secondary antibody solution
(in PBS) for 90 min (Supplementary Table 1). Sections were then
incubated with the avidin-biotin-peroxidase complex for 2 h (Vectastain
ABC kit, Vector Lab, Burlingame, CA, USA), and the immunoreactivity was
revealed by a peroxidase reaction using 3,3′-Diaminobenzidine
diaminobenzidine (DAB; Sigma) as the chromogen.
Quantification analysis: All analyses were done in the dorsal
striatum by an experimentally blind investigator. The quantification of
FosB, COX-2, astrocytes, microglia were carried out as in previous
studies (Bortolanza et al., 2015 a, b; Padovan et al., 2015). Digital
images were obtained using a Leica microscope
(Leica Microsystems Launches Leica
FW4000 - Cambridge, UK) under 20x or 40x objectives. The quantification
of the brain area was measured using the ImageJ system (National
Institutes of Health - NIH; Schneider et al., 2012).
Analysis of microglia (OX-42) and astrocyte (GFAP) immune labeling
morphology was conducted as described by Giocanti-Auregan et al. (2016)
using Fiji algorithms and a generated skeleton image. The parameters
analyzed were the number of branches, the number of intersections or
branching points, and the mean process length.
For FosB, COX-2, GFAP, and OX-42, the results were expressed as the
number of cells per 0.5 mm2. For GFAP and OX-42, the
results were also expressed as the number of branches of intersections
of branching points and process length per mm2 or
length per mm2.
Tissue collection for in situ fluorogenic reaction and gel
electrophoresis An independent experimental group of rats was used to
assess the in situ gelatinolytic activity and ROS concentration,
and another group for the substrate gel electrophoresis of
metalloproteinases and western blotting (see experimental
design ). At the end of the behavioral analyzes, animals were
anesthetized as described in the tissue collection and processingsection, immediately decapitated, and the brain removed.
To assess the in situ gelatinolytic activity of MMPs and ROS
presence, each brain was embedded in Tissue-tek®, quickly frozen in
isopentane (−40 °C, Sigma-Aldrich, St. Louis, MO, USA) and stored at −80
°C until histological processing. Coronal sections (5 µm) of the
striatum were cut using a freezing microtome (Leica, model CM1850).
To assess MMP-2 and MMP-9 presence by substrate gel zymography and the
MMP-3 protein concentration by western blot, the rat brain was removed
and dissected. The dorsal striata were micro-dissected on an ice-cooled
plate and immediately frozen in dry ice.
A block of tissue containing the SNc prevenient from the above described
experimental animal brain was post-fixed in buffered picric
acid-paraformaldehyde fixative for 6 h at 4 °C for TH-immunoreactivity
analysis (see immunohistochemistry ). The SNc blocks were quickly
frozen in isopentane (−40 °C) and stored at −80 °C. The brain sections
were collected and processed, as described in theimmunohistochemistry section.
Gelatinolytic zymography in situ: Zymography is a technique that
can be used for the detection of hydrolytic enzymes based on the
substrate repertoire of the enzyme. This technique allows for the
precise localization of the two MMPs identified as active
gelatin-degrading enzymes (gelatinases), i.e., gelatinase A (72-kDa
MMP-2) and gelatinase B (92-kDa MMP-9), in the gently fixed tissue
section (Galis et al., 1995; Lee et al. 2004; Nascimento et al. 2013). A
dye-quenched (DQ)-gelatin degradation assay to quantify MMP-2/MMP-9
activities was performed in the striatum tissue samples by zymography.In situ MMP activity was measured in 5 μm sections using
DQ-Gelatin (E12055, Molecular Probes, USA) as a fluorogenic substrate.
The gelatin with a fluorescent tag remains caged (no fluorescence) until
the gelatin is cleaved by MMPs 2/MMP-9. Sample sections were incubated
with 1.0 mg mL-1 of DQ-Gelatin in Tris-CaCl2 buffer
(50 mM Tris, 10 mM 177 CaCl2, 1 mM ZnCl2) in dark and humidified
chambers for 60 min. Proteolytic activity was detected as bright green
fluorescence, which indicated substrate breakdown. Arbitrary
fluorescence units were evaluated using ImageJ (NIH, USA). Negative
control sections were incubated in the same way as described above but
without DQ gelatin.
In situ reactive oxygen species measurement (ROS) In situvisualization of ROS production allows for the precise localization of
ROS in specific brain regions. The reaction was performed using
dihydroethidium [DHE, Sigma Chemical Co. (St. Louis, MO, USA)]. DHE
exhibits red fluorescence through interactions with superoxide and other
free radicals in the brain (Castro et al. 2009). Briefly, in situROS quantification was measured in sections 5-μm thick of the striatum
(obtained as described above for in situ gelatinolytic
zymography) incubated with DHE (10 μmol L-1; Castro et
al., 2009; Fernández et al. 2018) in dark wet chambers, at room
temperature, for 30 min. They were post-fixed in 4% paraformaldehyde in
0.1 M phosphate buffer for 10 min and then examined by fluorescence
microscopy (Leica Microsystems Launches Leica FW4000 - Cambridge, UK).
Bright red fluorescence represented ROS (arbitrary fluorescence units)
and was evaluated using ImageJ (NIH, USA). Negative control sections
were incubated in the same way as described above but without DHE.
Substrate gel electrophoresis for MMP-2 and MMP-9 and western blot
for MMP-3 Frozen striatum samples were homogenized in 300 mL extraction
buffer that consisted of 10 mM of CaCl2, 50 mM of
Tris-HCl, 1 mM of 1, 10 ortho-phenanthroline, phenylmethylsulphonyl
fluoride (PMSF, a serine protease inhibitor) at 100 µM and 1 mM of
N-ethylmaleimide to extract the proteins. Lesioned and non-lesioned
dorsal striata were processed separately. After 20 h at 4oC, the samples were centrifuged at 3000 g for 15 min,
and the protein content was measured using the Bradford (1976) method.
The samples were then diluted in the sample buffer (2% SDS, 125 mM
Tris–HCl pH 6.8, 10% glycerol, and 0.001% bromophenol blue) and
subjected to electrophoresis on 12% SDS-PAGE co-polymerized with 1%
gelatin (Sigma Chemical Co. St. Louis, MO, USA) as the substrate. After
electrophoresis was completed, the gel was incubated in a 2% Triton
X-100 solution followed by Tris-HCl buffer pH 7.4 + 10 mM CaCl2. The
gels were stained with 0.05% coomassie brilliant blue. The
gelatinolytic activity was detected as unstained bands against the
background of coomassie blue-stained gelatin. Metalloproteinases MMP-9
and MMP-2 protein in the striatum was detected as transparent bands
against a dark-blue background (Nascimento et al., 2013). Enzyme
activity was determined by densitometry using a Kodak Electrophoresis
Documentation and Analysis System (EDAS 290; Kodak, USA).
To analyze the antibody specificity by western immunoblotting, and to
quantify striatal MMP-3-related protein expression, total protein
extracts were obtained from the dorsal striatum, which was homogenized
in buffer containing 137 mM of NaCl, 20 mM of Tris, pH of 8.0, 1% NP-
40, 0.105% SDS, 10% glycerol, and protease inhibitors 0.2 mM of
phenylmethylsulphonyl fluoride (PMSF), 0.1 µM of aprotinin and 1 µM of
leupeptin (N-acetyl-L-leucyl-L-leucyl-L-argininal) and phosphatase
inhibitors (1 mM of NaF and 2 mM of
Na3VO4). After homogenization, the
sample was centrifuged (13,000 g at 4 °C for 20 min). The supernatant
and the precipitate were separated, and the total amount of protein in
the supernatant was determined by the Bradford (1976) method. Samples
containing 0.5 mg of protein were separated by polyacrylamide gel
electrophoresis containing 12% SDS-PAGE and electrically transferred to
a polyvinylidene difluoride (PVDF) membrane (Millipore, MA, USA).
Detection of the proteins on the membrane was done by sequential
incubations with the primary and secondary antibodies (see Table S1) and
evaluated using the ImmobilonTM Western Chemiluminescent HRP Substrate
detection system (Millipore) and registered by ImageQuant 350 detection
system (GE Healthcare).