Human Subjects and Tissue Processing
Brain tissue blocks containing the whole amygdala from adult normal human donors (n=15), were obtained from the Harvard Brain Tissue Resource Center (HBTRC) (). Blocks were dissected fresh from whole brains upon arrival at the HBTRC and immediately placed in 4% paraformaldehyde. The blocks were stored in 0.1 M phosphate buffer (PB) containing 4% paraformaldehyde for two weeks followed by one week in cryoprotectant solution (30% glycerol, 30% ethylene glycol, 0.1% NaAzide in PB). Blocks were then cut into sequential 2 mm coronal slabs using an antithetic tissue slicer and resectioned into 40μm serial sections on a freezing microtome (American Optical 860, Buffalo, NY). This method is well suited for measurements of volume and total cell numbers because it allows optimal tissue preservation, and avoids distortion and excessive variations of section thickness and tissue loss due to temperature changes that often occur while slicing a large cryoprotected tissue block. Incomplete sectioning due to alignment of the slab on the microtome amounted to a thickness of no more than 80–100 μm/slab. Sections were stored in the same cryoprotectant solution at −20 °C. Tissue from several regions from each brain was analyzed by a neuropathologist to determine any evidence of gross and/or macroscopic changes consistent with Alzheimer’s disease and other neurological illnesses, cerebrovascular accident, ethanol abuse, or other confounding factors.
Demographic and Descriptive Data of the Human Subject Cohort
Nonhuman Primate Subjects and Tissue Processing
Monkeys were given a lethal dose of sodium pentobarbital (100 mg/kg, i.p.) and were perfused intracardially with normal saline followed by a solution of 4% paraformaldehyde in 0.1 M phosphate buffer. The brains were removed, photographed, and placed in solutions of 10% (for 1 day) followed by 20% (for 4–5 days) glycerol plus 2% DMSO in 0.1 M phosphate buffer for cryoprotection. Then the brain was frozen in isopentane overnight and cut on a freezing microtome at 40 μm in the coronal plane. All sections were collected and saved in a solution for long-term storage consisting of 30% glycerol and 30% ethylene glycol in 0.1 M phosphate buffer.
Rat Tissue Processing
Male Sprague-Dawley rats (n=30 total; P240 n= 2; P120, n=5; P60, n=6; P45, n=5, P30, n=6, P15, n=6; Charles River Laboratories Inc., Wilmington, MA) were sacrificed by an overdose of sodium pentobarbital (130 mg/kg) and perfused with fixative (200 ml, 4% paraformaldheyde in 0.1 M PB). Brains were collected, postfixed (1 hr), cryoprotected (20% glycerol in 0.1 M PB overnight). A separate group of four rats (P60) was sacrificed by an overdose of sodium pentobarbital (130 mg/kg) but not perfused with fixative; after removal, the brains were postfixed for 1 week in 4% paraformaldehyde, then placed in cryoprotectant solution (30% glycerol, 30% ethylene glycol, 0.1% NaAzide in PB) until it sank to the bottom of the jar. Sections from all rat brains were serially sliced (30 μm) in coronal sections on a freezing microtome (American Optical 860, Buffalo, NY). Sets of serial sections including the whole rotrocaudal extent of the amygdala were used for histochemitry.
For human and monkey tissue, free-floating sections were incubated in citric acid buffer (0.1 M citric acid, 0.2 M Na2HPO4) heated to 80 °C for 30 minutes, then incubated in biotinylated WFA (1:500 μl; Vector Labs, Burlingame, CA) in 1% bovine albumine serum (BSA) for 24 hr at 4 °C. Sections were then incubated in streptavidin conjugated with horse radish peroxidase (1:5000 μl, Zymed, San Francisco, CA), and, finally, in nickel-enhanced diaminobenzidine/peroxidase reaction (0.02% diaminobenzidine, Sigma-Aldrich, 0.08% nickel-sulphate, 0.006% hydrogen peroxide) for visualization of the reaction product. Solutions for all the following steps were made in PBS with 0.5% Triton X (PBS-Tx). Each step was followed by washes in the same solution. Sections were counterstained with methyl green nuclear stain (Vector Labs, Burlingame, CA) and coverslipped. WFA histochemistry for rat tissue was identical to the one described above, with the exception that no antigen unmasking procedure was performed and buffers contained 0.2 % Tx.
For chondroitinase ABC digestion, following antigen unmasking with citric acid buffer, human amygdala sections were incubated overnight at 37 degrees °C in chondroitinase buffer consisting of 250 μl of chondroitinase ABC (10mU/μl, cat #C-2905, Sigma-Aldrich, St. Louis, MO) in 2ml of chondroitinase buffer (50 mM Tris, pH 8.0, 60 mM sodium acetate, and 0.02% BSA).
Dual Fluorescent Immunocytochemistry
A subset of five amygdalas from five different donors was used for dual fluorescent labeling. Free-floating sections were processed for antigen unmasking as above and then incubated in 5% BSA (1 hr) and placed in monoclonal primary antisera raised in mouse against glial fibrillary acidic protein (GFAP) (1:8,000 μl, G 3893, Sigma-Aldrich, St. Louis, MO) in 1%BSA at 4 degrees C for 72 hr. Immunoblotting assays show that this antibody is highly specific for GFAP and does not cross react with vimentin. Astrocytes, Bergmann glial cells, gliomas, and other glial cell derived tumors are specifically detected using this antibody (information provided by Sigma-Aldrich, St. Louis, MO). Sections were then incubated for 4 hr with Alexa Fluor horse anti-mouse (1:250 μl; wave length: 555) for 4 hr. Following several rinses, sections were placed in a solution containing biotinylated WFA (1:500 μl, Vector Labs, Burlingame, CA) in 1% BSA for 24 hr at 4 °C, followed by 4 hr incubation in Alexa Fluor Streptavidin (wave length: 488; 1:2000 μl). Solutions for all the steps above were made in PBS with 0.5% Triton X (PBS-Tx). Each step was followed by washes in the same solution. To eliminate lipofuscin autofluorescence, sections were incubated in 1mM CuSO4 for 10 minutes (Schnell, et al., 1999
). Sections were coverslipped with anti-fade Gel/Mount (Biomeda Corp., Foster City, CA). All fluorescent probes were purchased from Molecular Probes Inc. Eugene, OR.
Data Collection for Human Tissue
Four serial representative sections from each amygdala (section interval, 1040 μm), were labeled with WFA lectin histochemistry, counterstained with methyl green nuclear staining, and matched across subjects for rostral-caudal level according to morphological and cytoarchitectonic criteria as described previously (Pantazopoulos, et al., 2006
) (). A Zeiss Axioskop 2 Plus interfaced with StereoInvestigator 6.0 (Microbrightfield Inc., Willinston, VT) was used for analysis. The borders of each of the nuclei examined in this study, i.e. LN, BN, AB and CO, were delineated with a 1.6x objective according to well-established cytoarchitectonic criteria (Amaral, et al., 1992
, Gloor, 1997
, Sorvari, et al., 1995
). Sections were then scanned through the extent of the x, y, and z-axes using a 40x objective. On the basis of a previous knowledge (Bruckner, et al., 1994
, Hamidi, et al., 2004
, Pantazopoulos, et al., 2006
), the following criteria were established for classification of WFA-labeled objects. WFA-labeled glial cells are characterized by clearly distinguishable intracellular labeling and a small, round body surrounded by a multitude of short, thin and tightly packed (bush-like) processes (). WFA-labeled neurons show intracellular labeling and clear neuronal morphology (Luth, et al., 1992
). Perineuronal nets are typically distinguished easily from labeled cells, as they show neuron-like morphology, i.e. relatively large, polygonal or fusiform cell bodies and a small number of distinct primary dendrites, but the labeling has a hollow appearance, with a mesh-like, pericellular pattern ().
Quantitative analysis of double-labeled cells was performed using a Zeiss Axioscope 2 plus system equipped with FITC and TRITC filters and interfaced with Stereo-Investigator 6.0 image analysis software. Sections used for light microscopy WFA detection were adjacent to dual fluorescence labeled sections, and were used for tracing the borders of the LN, BN, ABN and CO on the latter (Amaral, et al., 1992
, Gloor, 1997
, Sorvari, et al., 1995
). Each nucleus was scanned systematically in its whole dorso-ventral and latero-medial extent and throughout the z-axis, using a 40x objective and fluorescent filters TRITC, 555 (GFAP) and FITC 488 (WFA). All WFA-labeled cells were recorded according to presence of WFA labeling alone or together with GFAP expression (). Co-localization was determined by overlaying the image captured using the TRITC filter and the FITC filter using Stereo-Investigator software.
Confocal microscopy was used to confirm data obtained with fluorescent microscopy. Approximately 20% of the WFA-labeled cells were re-sampled using a Leica Confocal Microscope System equipped with TRITC and FITC filters. This system allows scanning through the z-axis of each section in 1 μm increments, thus resolving among two or more neurons sharing the same Y and X (but not Z) coordinates. Co-localization of two markers was established when the respective fluorescence signal was clearly present at the same range within the z-axis for each marker examined and confirmed by a color change of overlapping markers (). Results obtained with confocal and fluorescence microscopy were virtually identical, confirming the validity of the latter.
Total number (Tn) of WFA-labeled, putatively CSPGs-positive, cells was calculated as Tn= i • Σn
, where Σn
= sum of the cells counted in each subject, and i
is the section interval (i.e. number of serial sections between each section and the next within each compartment, i.e. 26) as described in detail in previous work (Pantazopoulos, et al., 2006
). Stepwise linear regression models were used to analyze the effects of age, postmortem time interval (PMI), cause of death (acute vs chronic), hemisphere, and gender on total numbers of WFA-labeled cells and of percentages of WFA-labeled cells showing GFAP immunoreactivity using JMP v5.0.1a (SAS Institute Inc., Cary, NC). Microphotographs and figures were processed using Adobe Illustrator CS (Adobe Systems Inc., San Jose, CA). No adjustments were made to any aspect of the original photos except for size reduction.
Data Collection for Monkey and Rat Tissue
Sections from rhesus monkey and rat, containing the amygdala and stained with WFA lectin histochemistry were examined under light microscopy. Each amygdala nucleus was scanned systematically in its rostro-caudal, latero-medial and dorso-ventral extent. Each labeled element was examined and recorded according to morphological criteria and presence of intracellular labeling according to the same criteria described for human tissue.