Immunohistochemical analysis of the rat post-mortem tissue
Tissue preparation. Rats anaesthetized with tribromoethanol (1.5 g kg-1, Sigma-Aldrich, St. Louis, MO, USA) were euthanized 60 min after the last L-DOPA injection. The brain was fixed by transcardiac perfusion with 100 mL of Kreb’s-ringer buffer and 200 mL of buffered picric acid-paraformaldehyde fixative room temperature (Somogyi and Takagi, 1982 - 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). Brains were dissected and post-fixed in the same fixative for 60 minutes at 4 °C. The tissues were equilibrated with 30% sucrose in 0.1 M phosphate buffer and fully frozen. 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 throughout the rostrocaudal extent of the striatum (Bregma +2.76mm, Interaural 11.76mm – Bregma -2.28mm, Interaural 6.72mm) and the SNc (Bregma -4.44mm, Interaural 4.56mm – Bregma -6.24mm, Interaural 2.76mm) were cut (25 µm) using a freezing microtome (Leica, model CM1850).
Tyrosine hydroxylase (TH) Immunoperoxydase Labeling TH immunolabeling to confirm 6-OHDA lesion was detected following a standard peroxidase-based method (Gomes et al., 2008; Padovan-Neto et al., 2009). Sections were incubated overnight at room temperature with the primary antibody: rabbit anti-TH (1:4000, PelFreez, 01,229, USA for rats), followed by 2h of incubation with anti-rabbit biotinylated secondary antibody (1:250, Vectastain, Vector Laboratories, USA). Sections were then incubated with the avidin-biotin-peroxidase complex for 2 h (Vectastain ABC kit, Vector Lab, Burlingame, CA, USA). Immunoreactivity was revealed by a peroxidase reaction using 3,3′-Diaminobenzidine diaminobenzidine (DAB; Sigma-Aldrich, St. Louis, MO, USA) as the chromogen. The slices were mounted on slides and coverslipped for microscopic observations.
An in-depth analysis of NG2-glia by immunoconfocal morphometry . Immunofluorescent labeling of NG2-glia was achieved incubating brain sections in blocking buffer solution (3% normal goat serum, 0.05% Triton X-100 and 1% bovine serum albumin in phosphate-buffered saline) and then, overnight at 4 °C with a rabbit polyclonal antibody to NG2 (1:500; Millipore, Temecula, CA, USA). After thorough washing, sections were incubated for 90 min at room temperature in secondary antibodies conjugated to Alexa 488 against rabbit (fluorochrome, 1:1000 Invitrogen). The specificity of primary antibodies immunoreactivity was confirmed by the omission of the primary or secondary antibody and the verification of the absence of immunohistochemical staining in these sections. Brain sections were scanned and photographed by slide scanner (SCN400, Leica Microsystems Ltd., Mannheim, Germany) on Multi-channel confocal microscopy (Leica TCS-SP5, Leica Microsystems Ltd., Mannheim, Germany). Digital images were acquired for a wavelength of 488 nm (green), and the images were converted to TIFF format. Contrast levels adjusted using Adobe Photoshop v. 10.0 (Adobe Systems, San Jose, CA, USA).
Quantitative analysis of the cell number was carried out on the medial striatum dorsal and ventral areas (Bregma +0.96mm, Interaural 9.96mm – Bregma -0.24mm, Interaural 8.76mm - x20 objective) per animal, and results were expressed as an average density of NG2 glia in pixels. Only those on the surface of the sections, where antibody penetration was guaranteed, were counted. Images were digitized with a video camera (Leica DFC420), captured real magnification in grayscale, and evaluated with ImageJ (http:// rsb.info.nih.gov). Integrated optical density values (product of area and mean gray value) of corresponding sectors in each section were averaged. For this procedure, the stain’s mean gray value was expressed in arbitrary grayscale units where the scale ranges from 0 to 255 (0 representing the darkest, most intense labeling) to form one density measurement performed in the sections. After this, integrated optical density was calculated by multiplying the selected area with the mean gray value. Sampled areas consisted of regions of interest measuring 0.1 mm2. The average gray amount from an unstained area was subtracted from each section to correct for background immunoreactivity. All analyses were performed on the lesioned hemisphere of the brain (right) by an experimentally blind investigator.
Quantitative analysis of the ramification index, number of branches, number of junctions, average branch length, and soma area of NG2-glia was carried out on the medial striatum dorsal and ventral areas (Bregma +0.96mm, Interaural 9.96mm – Bregma -0.24mm, Interaural 8.76mm- x40 objective), according to previous studies (Heppner et al., 1998; Eder et al., 1999). Either adjustment to contrast or brightness were made uniformly to all parts of the image. A Sholl analysis method was developed to quantify NG2-glia morphology in immunofluorescent images of the striatum, according to the following: the resulting images were converted to a binary signal and analyzed using the Simple Neurite Tracer plugin of the Fiji software, ImageJ (https://imagej.net/Fiji/Downloads). Cellular branches were manually traced and, the center for the Sholl analysis was pointed at the centroid of the nucleus. Concentric circles were automatically drawn, beginning at 2 μm from the center and increasing 2 μm with every circle. The Sholl analysis plugin was then applied to all traced cells to collect data on the intersections between branches and each increasing circle to create a Sholl plot. For each rat, a mean Sholl plot was generated. The ramification index (RI) was calculated using the following formula: RI = cell area/convex area, where “convex area” is the area of a polygonal object defined by the cells’ most prominent projections (Schilling and Eder, 2015). The length of NG2-glia processes was defined as the distance between the soma and the detectable end of an extended process identified by NG2 staining. NG2-glia diameter (soma area) was determined by measuring the cell body’s longest axis through the nucleus (um2).
Dual labeling of NG2-glia and glial fibrillary acidic protein (GFAP, to reveal astrocytes) or OX-42 (CD11b/c equivalent protein of microglia to reveal microglia) was achieved incubating brain sections in blocking buffer solution (3% normal goat serum, 0.05% Triton X-100 and 1% bovine serum albumin in phosphate-buffered saline) and then, overnight at 4 °C with a rabbit polyclonal antibody to NG2 (1:500; Millipore, Temecula, CA, USA) and mouse monoclonal antibody to GFAP (1:700; Millipore, Temecula, CA, USA) or rabbit polyclonal antibody to NG2 and mouse monoclonal antibody to OX-42 (1:600; Abcam, Branford, CT, USA). After thorough washing, sections were incubated for 90 min at RT in secondary antibodies conjugated to Alexa 488 against rabbit and Alexa 568 against mouse (fluorochromes, 1:1000 Invitrogen). The specificity of primary antibodies immunoreactivity was confirmed by the omission of the primary or secondary antibody and the verification of the absence of immunohistochemical staining in these sections. Immunofluorescent digital images were obtained using a Leica microscope (Leica Microsystems Launches Leica FW4000 - Cambridge, UK), and were acquired separately for each wavelength [488 nm (green) and 568 nm (red)] and then merged. The striatal area’s quantification was measured using the ImageJ system (National Institutes of Health - NIH; Schneider et al., 2012). Striatal NG2, GFAP, and OX-42 immunolabeled cells were quantified at 20X magnification. The striatum was unilaterally evaluated in the dorsolateral, dorsomedial, ventrolateral, and ventromedial quadrants. According to the coordinates of the Franklin and Paxinos (1997) rat brain atlas, the regions were located. For double labeling, the results of NG2, GFAP, and OX-42 were analyzed as the number of cells per 0.5 mm2, and they were expressed in percentage of double labeling per total of NG2 cells.