SUPPLEMENTARY MATERIALS AND METHODS
Animals
For in vivo experiments, male Wistar Hannover (WH) rats and Spontaneuosly
Hypertensive Rats (SHR) (Taconic Farms, Germantown, NY) were kept three per cage with free
access to water and a standard diet at 22°C under a 12:12 hour dark-light cycle. For in vitro
studies, three-week-old WH rat males were used for isolation of brain microvascular endothelial
cells; eigth-day-old Sprague Dawley rat pups (Taconic Farms) were used for isolation of
cerebellar granule cells; and one-day-old Sprague Dawley rat pups were used for isolation of
cortical microglial cells. The National Institute of Mental Health Animal Care and Use
Committee (Bethesda, MD, USA) approved all procedures. All efforts were made to minimize
the number of animals used and their suffering (National Institutes of Health Guide for the Care
and Use of Laboratory Animals, Publication No. 80-23, revised 1996).
In vivo studies
Short-term AT1 receptor blockade. To study the effects of short-term AT1 receptor
blockade on LPS-induced immune response, 9-week-old WH rats received daily subcutaneous
injection of vehicle or AT1 receptor blocker candesartan (CV-11974, Astra-Zeneca, Mölndal,
Sweden) at the dose of 1 mg/kg/day for three consecutive days. Candesartan was initially
dissolved in 0.1 N Na2CO3 and further diluted to working concentration with isotonic saline, at a
final pH of 7.5–8.0. The animals were injected intraperitoneally with 50 µg/kg
1
lipopolysaccharide (LPS, Escherichia coli serotype 055:B5; Sigma-Aldrich, St Louis, MO) or
sterile saline on day 3 and were euthanized three hours later by fast decapitation for plasma and
tissue collection.
Behavioral studies. To establish the effect of short-term AT1 receptor blockade on LPSinduced anorexia and body weight loss, 9-week-old WH rats were housed individually with free
access to a standard diet and water. The rats were injected daily with either candesartan (1
mg/kg/day sc.) or vehicle until the end of experiment. On the third day, the rats were injected
with LPS (50 µg/kg ip.) or saline and were euthanized 2 days later. Body weight and food
consumption were monitored daily throughout the experiment.
For determination of anxiety, 9-week-old SHR were treated subcutaneously with 1 mg/kg
body weight candesartan or vehicle for three consecutive days followed by intraperitoneal
injection of LPS, 50 µg/kg body weight. Three hours after LPS injection, the rats were tested for
anxiety-like behavior on an elevated plus maze (Columbus Instruments, Columbus, OH). The
elevated plus maze consisted of two opposite open arms (50x10 cm) and two opposite closed
arms of the same dimensions with walls 40 cm high. The arms were connected through a central
square (10x10 cm) forming the shape of cross and elevated 50 cm above the ground. Testing was
conducted in a quiet room under dim illumination. To facilitate adaptation, the animals were
placed in the testing room at least 1 hour prior to testing. The test began by placing the rat on the
central square facing an open arm. The time spent and the number of entries into the open and
closed arms were recorded during 5 min. The arm entry was defined by placing the all four limbs
on the arm (Saavedra et al., 2006).
2
Sustained AT1 receptor blockade. To determine the effects of sustained AT1 receptor
blockade, 9-week-old WH rats were anesthetized with 30 mg/kg pentobarbital, and were
implanted subcutaneously with osmotic minipumps (model 2002, Alza Scientific Products, Palo
Alto, CA). Half of the animals received minipumps containing candesartan diluted as above to be
delivered at the rate of 1 mg/kg/day; the rest of animals received minipumps filled with vehicle.
On day 14, the animals were injected with LPS (50 µg/kg ip.) or sterile saline and were
euthanized three hours later by fast decapitation for plasma and tissue collection. For
immunohistochemical studies, separate groups of animals treated as above were anesthetized
with 50 mg/kg pentobarbital three hours after LPS or saline injection and were transcardially
perfused with saline followed by a fixative solution containing 4% formaldehyde (Mallinckrodt
Chemicals, Philipsburg, NJ) in phosphate-buffered saline (PBS: 5.6 mM Na2HPO4, 1.0 mM
KH2PO4, 154 mM NaCl, pH 7.4). After perfusion, the brains were dissected, post-fixed in the
same fixative overnight at 4°C and cryoprotected by consecutive immersions into 10, 20, and
30% sucrose in PBS at 4°C until saturation. Brains were frozen in isopentane on dry ice and
stored at −80 °C until used.
Plasma and tissue collection. Trunk blood was collected into pre-chilled tubes containing
1 mg/ml EDTA (Sigma-Aldrich), centrifuged at 1,800 g for 15 min at 4ºC and the plasma was
stored at -80ºC until used. The brains were quickly dissected, frozen in isopentane on dry ice and
stored at -80°C until used.
In vitro studies
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Cerebellar granule cell (CGC) culture. CGCs were isolated from eight-day-old Sprague
Dawley rat pups as described previously (Gao, et al 1995). Briefly, 10 cerebella from rat pups
were broken down into small pieces by repeated pipetting and incubated with 0.25 mg/ml trypsin
(Sigma-Aldrich) in neurobasal medium (Invitrogen, Carlsbad, CA) for 12 min at 37°C. The
digest was briefly treated with 0.52 mg/ml trypsin inhibitor (Sigma-Aldrich) and 0.08 mg/ml
DNase II (Sigma-Aldrich) and pelleted by centrifugation. The cells were resuspended in
B27/neurobasal medium (Invitrogen), plated at a density of 1.2 × 106 cells/ml onto poly-L-lysine
coated 6-well plates (Becton Dickinson and Company, Franklin Lakes, NJ) and cultured for 6 to
14 days at 37°C under 5% CO2 atmosphere. We have confirmed that contaminating glial cells
represented only 2-3% of the total cell population as initially described (Gao, et al 1995). Before
the experiment, the cells were starved overnight in plain neurobasal medium and were
subsequently treated for 4 h with 500 ng/ml LPS with or without pre-treatment with 5 μM
candesartan for 2 h. At the end of the experiment the medium was saved and the cells were
rinsed with PBS and lysed with TRIzol reagent (Invitrogen) for RNA isolation as described
below. Candesartan was initially prepared as 10 mM stock in DMSO and further diluted in
incubation medium to 0.05% final DMSO concentration. The control cells were treated with
vehicle (0.05% DMSO).
Brain microvascular endothelial cell culture. Primary endothelial cell cultures were
prepared from 3-week old rats under aseptic conditions as described previously (Nakagawa, et al
2009) with a few modifications. Forebrains cleaned of meninges and white matter were minced
into small pieces and dissociated by 25 up-down strokes with a 5 ml pipette in Dulbecco’s
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modified Eagle’s medium (DMEM, Sigma-Aldrich) containing 1 mg/ml collagenase type 2
(Worthington Biochemical Corp., Lakewood, NJ), 15 µg/ml DNase I (Sigma-Aldrich), and 50
µg/ml gentamycin (Mediatech, Herndon, VA) followed by digestion for 90 min at 37°C under
constant shaking. The cells were sedimented by centrifugation at 1000g for 10 min at 4°C,
resuspended in DMEM containing 20% bovine serum albumin (BSA, Sigma-Aldrich) and
recentrifuged at 1000g for 20 min at 8°C. The pellet was resuspended in DMEM containing 1
mg/ml collagenase-dispase (Roche Applied Sciences, Basel, Switzerland) and 6.7 µg/ml DNase I
(Sigma-Aldrich) and further digested for 45 min at 37°C. The dissociated cells were sedimented
at 800g for 8 min at 8°C, washed, recentrifuged and plated on 35 mm plastic dishes coated with
collagen type IV and fibronectin (both 0.1 mg/ml, Sigma-Aldrich) at 4x 105 cells/ml. The cells
were kept in DMEM/F12 (Sigma-Aldrich) supplemented with 10% plasma-derived serum
(Animal Technologies Inc., Tyler, TX), 1.5 ng/ml basic fibroblast growth factor (Roche Applied
Sciences), 100 µg/ml heparin (Sigma-Aldrich), 5 µg/ml insulin, 5 µg/ml transferrin, 5 ng/ml
sodium selenite (insulin-transferrin-sodium selenite media supplement, Sigma-Aldrich), 50
µg/ml gentamycin and 4 µg/ml puromycin (Sigma-Aldrich) at 37°C in a humidified 5% CO2
atmosphere. On the third day the medium was replaced with puromycin-free medium
supplemented with 500 nM hydrocortisone (Sigma-Aldrich). After the first passage, the cells
were plated onto collagen IV- and fibronectin-coated 6-well plates at 4x105 cells/ml and used for
experiments when they reached 80% confluence. Before the experiment, the cells were starved
overnight in serum-free plain DMEM/F12 medium and were subsequently treated with 100
ng/ml LPS for 4 h with or without prior pre-incubation with 10 µM candesartan for 2 h. At the
end of incubation, the medium was saved and the cells were washed in PBS and lysed in Trizol
reagent for RNA isolation, as described below.
5
Cortical microglial cell culture. Microglia was prepared from one-day-old Sprague
Dawley rat pups as initially described by Giulian and Baker (1986) with slight modifications.
Pups were decapitated and the whole brain was placed into Hanks’ Balanced Salt Solution
(Invitrogen) containing 10 mM HEPES and 1 mM sodium pyruvate. Cerebral cortices were
detached and minced by scissors, followed by 0.25% trypsin (Invitrogen) digestion for 5 min at
37°C. Cortices were transferred to fresh tubes and dissociated by trituration in a complete culture
medium DMEM/F12 (Invitrogen), supplemented with 10% FBS (Invitrogen), 100 U/ml
penicillin-streptomycin (Invitrogen), 0.1 mg/ml DNase I (Sigma-Aldrich). After centrifugation at
1000 rpm for 5 min, mixed cells were resuspended in the complete medium and passed through a
70 μm cell restrainer (BD Falcon, Vernon Hills, IL). The cell suspension was plated at a density
of 2 brains/185 cm2 flask in a humidified 5% CO2 atmosphere at 37°C. The medium was
replaced with fresh one after 24 h and then twice per week. After 10 days of culture, flasks were
shaken for 2 h at 200 rpm on a rotary shaker at 37°C to detach the microglia. Floating cells were
collected by centrifugation at 1000 rpm for 5 min and then seeded onto 6-well plates in a fresh
complete medium at a density of 2x106 cells/well. Microglia cultures were washed 30 min after
seeding to remove non-adherent cells. Cells were allowed to grow for 24 h at 37°C in a
humidified atmosphere enriched with 5% CO2 and then starved overnight in plain DMEM/F12
medium. Subsequently, the cells were treated for 4 h with 100 ng/ml LPS with or without 2 h
pre-incubation with 10 μM candesartan. At the end of experiment the medium was saved and the
cells were rinsed with PBS and lysed in TRIzol reagent (Invitrogen) for RNA isolation as
described below.
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Determination of cytokine levels
Cytokines in plasma and cell culture medium were measured by commercial enzymelinked immunosorbent assay (ELISA) kits from BioSource (Camarillo, CA) for IL-6 and TNF-α
and from R&D Systems (Minneapolis, MN) for IL-1β and IL-10.
Microdissection of brain structures.
Three hundred µm thick coronal sections from fresh-frozen brains were cut on a Leica
CM3050S cryostat (Leica Microsystems, Wetzler, Germany) at -10°C, mounted on Superfrost
Plus slides (Daigger, Vernon Hills, IL) and immediately frozen on dry ice and stored at -80°C
until further processed. Sections were used for punch microdissection as follows: three sections
between -1.3 and -2.2 mm from bregma for the PVN and the central nucleus of the amygdala;
two sections between -0.7 and -1.3 mm from bregma for the SFO; three sections between -2.2
and -3.1 mm from bregma for the CA1 subdivision of the hippocampus; and three sections
between 3.7 and 2.9 mm from bregma for the ventromedial prefrontal cortex (Paxinos and
Watson, 1998). Punch microdissection was performed under stereomicroscope control using
Harris Uni-Core microdissection needles (Electron Microscopy Sciences, Hatfield, PA), 0.75
mm diameter for the PVN and 1 mm for the other brain structures. The individual punches
dissected from same nucleus were pooled and transferred into ice-cold micro-homogenizers
(Kontes Glass Co., Vineland, NJ) and immediately homogenized in TRIzol reagent for RNA
isolation as described below. Tissues from each rat were studied individually.
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Determination of mRNA expression of pro-inflammatory factors by real-time PCR
RNA from microdissected brain tissue or cell culture lysates was isolated by TRIzol
reagent (Invitrogen) followed by purification using the RNeasy Mini kit (Qiagen, Valencia, CA)
according to the manufacturer’s instructions. Total RNA (0.15 μg from brain tissue; 1 μg from
cultured cells) was reversely transcribed into cDNA using Super-Script ΙΙΙ first-Strand Synthesis
kit (Invitrogen) according to the manufacturer's protocol and quantified by real-time PCR using
the DNA Engine Opticon system (Bio-Rad, Hercules, CA). The reaction consisted of 10 µl
SYBR Green PCR Master Mix (Applied Biosystems, Foster City, CA), 2 µl of diluted cDNA
and 0.3 µM primers in a total volume of 20 µl. Primers used in the study are listed in
supplementary Table S1. Amplification was initiated by denaturation at 95°C for 10 min,
followed by 40-50 cycles of 95°C for 15 sec and 60°C for 60 sec. At the end of amplification, the
specificity of the PCR products was confirmed by melting point analysis. Serial dilutions of rat
cDNA from the same source as the samples were used to obtain a standard curve. The target
cDNA from individual samples was quantified by determining the cycle threshold and
comparison with standard curves. The relative amount of the target cDNA was normalized to
GAPDH mRNA and expressed as a fold change relative to vehicle-saline group. The expression
of GAPDH mRNA was not affected by any of the treatments used.
Angiotensin II receptor autoradiography
Fresh-frozen brains were cut to 16 μm thick coronal sections containing PVN and SFO
on a cryostat at -20°C. The sections were thaw-mounted on Superfrost Plus slides, vacuum-dried
8
overnight at 4°C and stored at −80°C until used. The receptor autoradiography was performed as
described previously (Tsutsumi and Saavedra, 1991). Briefly, the sections were preincubated for
15 min at 22°C in a 10 mM sodium phosphate buffer, pH 7.4, containing 0.005% bacitracin
(Sigma-Aldrich), 5 mM Na2EDTA, 120 mM NaCl and 0.2% proteinase-free BSA (SigmaAldrich). Sections were then incubated for 2 h at 22°C in fresh buffer containing 0.5 nM
[125I]Sar1-Ang II. To characterize Ang II receptor subtypes, adjacent tissue sections were
incubated as above in the presence of either 10 μM losartan (DuPont Merck, Wilmington, DE), a
selective AT1 receptor antagonist, or 5 μM unlabeled Ang II (Peninsula Laboratories) to assess
nonspecific binding. The number of AT1 receptors was determined as the specific binding
displaced by losartan. After incubation, slides were washed four times for 1 min in Tris–HCl, pH
7.4 at 4°C, followed by a 30 s rinse in distilled water at 4°C. Slides were then dried under a
stream of cold air and exposed to Biomax MR film (Eastman Kodak, Rochester, NY). Optical
densities of autoradiograms were normalized after comparison with [14C]-microscale standards
as described (Tsutsumi and Saavedra, 1991) and quantified by computerized densitometry using
the Scion Image 4.0.2 software (Scion Corporation, Frederick, MD) based on the NIH Image
Program of the National Institutes of Health. Binding signal was expressed as fmol/mg of protein.
Localization and quantification of mRNA expression by in situ hybridization
Fresh-frozen brains were cut on a cryostat at -20°C to 16 µm thick coronal sections
containing PVN, SFO and choroid plexus. The sections were thaw-mounted on Superfrost Plus
slides, dried for 5 min at 50°C and stored at -80°C until used.
9
In situ hybridization of c-Fos and IL-1β. In situ hybridization of c-Fos and IL-1β mRNA
was performed using [35S]-labeled synthetic oligonucleotide probes. Antisense probes for the rat
IL-1β 5’-CCACGGGCAAGACATAGGTAGCTGCCACAGCTTCTCCACAGCCAC-3’ (Kim,
et al 2004) and c-fos 5’-GCAGCGGGAGGATGACGCCTCGTAGTCCGCGTTGAAACCC
GAGAA-3’ (Vaisanen, et al 2004) and their corresponding sense probes were synthesized by
Bioserve Biotechnologies (Beltsville, MD). Labeling was performed using the terminal
deoxynucleotidyl transferase (New England Biolabs, Ipswich, MA) to a specific activity of 3–4 ×
108 cpm/μg. Each reaction was performed with 10 pmol of oligonucleotide probe in the presence
of 200 μCi of [α-35S] ATP (NEN/Perkin Elmer, Waltham, MA). The labeled oligonucleotides
were separated from unincorporated nucleotides using ProbeQuant G-50 Micro columns (GE
Healthcare, Buckinghamshire, UK). The labeling of oligoprobes was monitored with liquid
scintillation counting. Prior to use, the sections on slides were warmed in a desiccator at room
temperature and then fixed in 4% formaldehyde (Mallinckrodt Chemicals) in PBS for 10 min.
After two washes in PBS, they were acetylated for 10 min in 0.1 M triethanolamine HCl, 0.9%
NaCl, and 0.25% acetic anhydride (Sigma-Aldrich), delipidated in ethanol and chloroform and
air-dried. The sections were covered with 150 µl hybridization buffer containing 50% formamide
(Sigma-Aldrich), 1×Denhardt’s solution (Sigma-Aldrich), 10% dextran sulfate (Oncor Inc,
Gaithersburg, MD), 100μg/ml PolyA (Sigma-Aldrich), 100μg/ml salmon testes DNA (SigmaAldrich), 100μg/ml yeast RNA (Sigma-Aldrich), 250μg/ml yeast tRNA (Invitrogen), 150mM
DTT (MP Biomedicals), 0.2% sodium dodecyl sulfate (SDS) (Quality Biological Inc,
Gaithersburg, MD), 0.2% sodium thiosulfate (Sigma-Aldrich) and 2 × 107cpm/ml sense or
antisense probe and the slides were covered with glass cover slips. After hybridization for 18 h at
40°C in humidified chamber, the coverslips were removed by rinse in 4× standard saline citrate
10
(SSC) and the sections were washed 3 x 5min in 1x SSC at room temperature followed by 15
min high-stringency washings consecutively in 1x SSC, 0.5x SSC and 0.2x SSC at 55°C. The
sections were dehydrated in graded ethanols containing 0.3 M ammonium acetate, air dried and
exposed to Biomax MR film (Eastman Kodak). The optical densities of autoradiograms were
measured using the Scion Image 4.0.2 software (Scion Corporation). The intensities of the
hybridization signals were expressed as nCi/g tissue equivalent after calibration with [ 14C]microscales standards (Tsutsumi and Saavedra, 1991), and after subtraction of the non-specific
hybridization signal obtained in the same areas of adjacent sections hybridized with sense
(control) probes.
In situ hybridization of AT1A receptor. In situ hybridization was performed using [35S]labeled antisense and sense (control) riboprobes as described previously (Jőhren, et al 1995).
Fragments from the 3’-non-coding regions of the AT1A cDNA were subcloned into the polylinker
site of the pBluescripts KS(+) vector (Stratagene, La Jolla, CA). A 368 bp PalI fragment of the
rat AT1A cDNA was ligated into the EcoRV site of the pBluescript vector. The orientation of the
insert was determined by restriction analysis with AvaII, which cuts asymmetrically within the
subcloned AT1A cDNA fragment. The subclones containing plasmids were linearized with
HindIII or EcoRI to generate [35S]-labeled sense and antisense probes. Radiolabeled probes were
prepared by in vitro transcription in the presence of [35S]-UTP (MP Biomedicals, LLC, Solon,
OH), 1μg of linearized subclone plasmid and T7 or T3 RNA polymerase, using a MAXI-Script®
T7/T3 kit (Ambion, Austin, TX) according to the protocol of the manufacturer. After
transcription, the template DNA was digested with DNase I for 15 min at 37 °C. Unincorporated
nucleotides were removed by centrifugation through ProbeQuant™ G-50 Micro Columns (GE
11
Healthcare). The labeling of riboprobes was monitored with liquid scintillation counting and the
specificity verified by a preliminary experiment with the PVN as a positive control (Jőhren, et al
1995). Prior to use, the sections prepared for hybridization were warmed in a desiccator at room
temperature and then fixed in 4% formaldehyde in PBS for 10 min. After two washes in PBS,
they were acetylated for 10 min in 0.1 M triethanolamine HCl, 0.9% NaCl, and 0.25% acetic
anhydride, delipidated in ethanol and chloroform, and air-dried. The sections were covered with
150μl hybridization buffer containing 50% formamide, 0.3 M NaCl, 1 mM EDTA, 20 mM Tris,
pH 7.5, 1x Denhardt's solution, 10% dextran sulfate, 100μg/ml salmon testes DNA, 250 μg/ml
yeast RNA, 250 μg/ml yeast tRNA, 150 mM DTT, 0.2% SDS, 0.2% sodium tiosulfate, and
2×107 cpm/ml sense or antisense probe, and the slides were covered with glass cover slips. After
hybridization for 18 h at 54 °C, coverslips were removed by rinse in 4x SSC and the sections
were washed 3 x 5 min in 4x SSC at room temperature. Non-hybridized probes were digested by
incubation with 40μg/ml RNase A (Sigma-Aldrich) for 30 min at 37 °C followed by high
stringency wash in 0.1X SSC at 65 °C for 60 min. The sections were dehydrated in graded
ethanols containing 0.3 M ammonium acetate, air dried and exposed to Biomax MR film (Kodak,
Rochester, NY). The mRNA expression was analyzed by measuring optical film densities as
described above. Two consecutive sections per rat were averaged and used to calculate group
means. Each animal was quantified independently. Results are presented as means ± SEM.
Tissue background was not different from the film background and no hybridization signal over
background was observed using sense RNA probes.
Immunohistochemical localization and quantification of c-Fos protein and microglia
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Formaldehyde-fixed rat brains were cut on a cryostat at -20°C to 16 μm thick coronal
sections containing PVN. The sections were thaw-mounted to Superfrost Plus slides, vacuumdried overnight at 4°C and stored at -80°C until used. Before the immunohistochemical staining,
the slides were warmed in a desiccator at room temperature, immersed in ice-cold acetone for 15
min, and incubated 3 x 5 min in Tris-buffered saline (TBS: 20 mM Tris, 136 mM NaCl, pH 7.4)
containing 0.2% Triton X-100 (Sigma-Aldrich). Intracellular peroxidase was quenched with
0.3% hydrogen peroxide containing 0.1% sodium azide in TBS for 30 min at room temperature.
The sections were washed 3 x 5 min in TBS and blocked for 60 min with TBS containing 1%
protease-free BSA, 10% normal goat serum (Vector Laboratories, Burlingame, CA), and 4
drops/ml of avidin solution (Avidin/Biotin Blocking Kit, Vector Laboratories). After blocking,
the slides were rinsed briefly in TBS and incubated overnight at 4°C with a primary antibody
diluted in TBS containing 1% BSA, 5% normal goat serum and 4 drops/ml biotin (Avidin/Biotin
Blocking Kit, Vector Laboratories). Rabbit polyclonal antibody against rat c-Fos (Ab-5, 1:10000,
Calbiochem, La Jolla, CA), or mouse monoclonal antibody against rat CD11b/c (Ox-42, 1:300,
BD Pharmingen, San Jose, CA) were used as primary antibodies. The sections were washed 4 x
5 min with TBS containing 0.05% Tween-20 (TBS-T) and incubated for 2 h at room temperature
with the goat biotin-conjugated secondary antibody against rabbit or mouse IgG (1:1000,
Jackson ImmunoResearch, West Grove, PA) diluted in TBS containing 1% BSA and 5% normal
goat serum. The sections were then washed 4 x 5 min with TBS-T and incubated 30 min with
avidin and biotin-conjugated horseradish peroxidase H (Vectastain ABC kit, Vector
Laboratories). The sections were exposed to DAB reagent (Vector Laboratories), counter-stained
with hematoxylin, dehydrated with ethanol of increasing grade (from 50 to 100%), cleaned with
13
xylene and mounted with coverslips using xylene-based mounting medium (Permount, Fisher
Scientific, Fair Lawn, NJ).
Data acquisition and quantification. For data acquisition and quantification, sections
were examined by an investigator blinded to the protocol used and the identity of the slides,
using a Zeiss Axioscope light microscope (Zeiss, Jena, Germany) and the images were acquired
by a Zeiss Axiocam digital camera under the constant conditions of light illumination. Induction
of c-Fos protein in the PVN was evaluated by automated image analysis using Image J software
(National Institutes of Health, Bethesda, MD). The images analyzed were converted into grey
scale and the identical threshold was applied to all pictures to eliminate background. Positive
cells were counted automatically by particle analysis and data were expressed as the number of
c-Fos-positive cells per section of PVN. Five to eight animals per group were analyzed
individually. Microglia activation was evaluated as an area of PVN positive for the microglia
marker Ox-42 staining (Milligan, et al 1991). The analysis was performed by an automated
procedure using Image J software (National Institutes of Health). Immunohistochemical images
were
processed
by
color
deconvolution
plug-in
using
H-DAB
vector
(http://rsbweb.nih.gov/ij/plugins) (Ruifrok and Johnston, 2001) to separate microglia staining
from hematoxylin counter-staining. The images of microglia were then subjected to the same
threshold to eliminate background and converted into black and white scale. The resulting
images were analyzed automatically by particle analysis function and data are expressed as a
proportion of Ox-42 positive area to the total area of the PVN. The increase in the proportion of
the area occupied by microglia was used as a measure of activation as reported previously
(Popovich, et al 1997).
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Statistical analysis
All results are expressed as means ± SEM for groups of 5-10 animals measured
individually. Results from cell culture experiments are expressed as means ± SEM of three
different cell preparations assayed individually. . To address the differences among all individual
groups, we analyzed the data by one-way ANOVA followed by the post hoc Newman-Keuls’
test for all determinations with significant omnibus ANOVA. P<0.05 was considered as
statistically significant. All statistics were performed with the use of GraphPad Prism 5.02
software (GraphPad Software Inc., San Diego, CA).
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