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).