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.