Supplementary Materials and Methods
The present study was conducted in accordance with the Guiding Principles for the
Care and Use of Laboratory Animals, as adopted by the Committee on Animal
Research of Hoshi University, which is accredited by the Ministry of Education,
Culture, Sports, Science and Technology of Japan.
Every effort was made to
minimize the numbers and any suffering of animals used in the following experiments.
Animals were used only once in the present study.
All behavioral experiments were
conducted in a single-blind fashion to avoid the effect of subjectivity.
Animals
All animals were housed at a room temperature of 22 ± 1˚C with a 12-h light-dark
cycle (light on 8:00 am to 8:00 pm).
Food and water were available ad libitum.
Intrathecal injection
Intrathecal (i.t.) injection was performed as described by Hylden and Wilcox (1980)1
using a 25-l Hamilton syringe (Hamilton Company, NV, USA) with a 30 1/2-gauge
needle. The needle was inserted into the intervertebral space of the spinal cord of
unanesthetized mice.
A reflexive flick of the tail was considered to indicate the
accuracy of each injection.
The volume for intrathecal injection was 4 l.
Neuropathic pain model
Mice were anesthetized with 3% isoflurane. We produced a partial sciatic nerve
ligation model by tying a tight ligature with 8-0 silk suture around approximately 1/3 to
1/2 the diameter of the sciatic nerve on the right side (ipsilateral side) of mice under a
light microscope (SD30, Olympus, Tokyo, Japan), as described previously2.
In
sham-operated mice, the nerve was exposed without ligation.
Gene transcription profiling using the spinal cord of nerve-ligated and
sham-operated mice
In the present study, samples were prepared 7 days after partial sciatic nerve ligation
or sham operation as a control. For sample preparation, the ipsilateral side of the mouse
spinal cord was quickly removed after decapitation and rapidly transferred to a tube
filled with RNA stabilization reagent (RNAlater; QIAGEN, Heiden, Germany). Total
RNA of the spinal cord was prepared using an RNeasy Lipid Tissue Mini Kit
(QIAGEN) according to the manufacturer’s instructions. The RNA concentration was
measured at 260 nm with a NanoDrop ND-1000 UV-Vis spectrophotometer (NanoDrop
Technologies Inc., Rockland, DE, USA). RNA was subjected to quality analysis on a
2100 Bioanalyzer (Agilent Technologies, Palo Alto, CA, USA) using an RNA 6000
NanoChip. For microarray-based expression analysis, cRNA labeled with cyanine 3 was
synthesized from 500 ng of total RNA using a low RNA input fluorescent linear
amplification kit (Agilent Technologies). cRNA quantity and quality were measured
using a NanoDrop and 2100 Bioanalyzer. Aliquots of 1.65 g of fragmented cRNA
were hybridized to G4122F whole mouse genome microarrays (Agilent Technologies)
following the standard protocol (version 4.0) provided by the manufacturer. The slides
were scanned with a DNA microarray scanner (Agilent Technologies). Scanned images
were analyzed with Feature Extraction software (Agilent Technologies). The obtained
expression data were imported into GeneSpring GX 7.0 software (Agilent
Technologies) for normalization and data analysis. For per chip normalization, the 50 th
percentile of the signal values taken from each microarray was used as the normalizing
reference. Per gene normalization was based on calculating the fold expression levels
relative to the median in sham-operated mice (n = 3). The fold change in gene
expression was calculated as follows: sciatic nerve ligation/sham operation.
Tissue dissection and preparation of the cytosol fraction
Tissue dissection and preparation of the cytosol fraction were performed following
the methods described previously3.
1, 3, 5, 7, 14 and 28 days after the surgery, the
lumbar spinal cord after nerve ligation or sham operation was quickly removed by
decapitation and dissected on an ice-cold metal plate. The tissue was homogenized in
10 volumes of ice-cold buffer containing 20 mM Tris–HCl (pH 7.5), 2 mM ethylene
diamine tetraacetic acid (EDTA), 0.5 mM ethylene glycol tetraacetic acid (EGTA), 1
mM phenylmethylsulfonyl fluoride, 25 g of leupeptin per ml, and 10 g of aprotinin
per mL, 10 mM Sodium fluoride, 1 mM sodium orthovanadate, and 0.32 M sucrose.
The homogenate was centrifuged at 1000 x g for 10 min at 4 °C and the supernatant was
ultracentrifuged at 100,000 x g for 30 min at 4°C. The resulting supernatant was
retained as the cytosolic fraction.
Western blotting
The protein levels were performed following the methods described previously3,4.
An aliquot of tissue sample was diluted with an equal volume of 2 x electrophoresis
sample buffer (EZ Apply, Atto Co., Tokyo, Japan). Proteins were separated by size on
5-20% sodium dodecyl sulfate (SDS)-polyacrylamide gradient gel or 15% tris
SDS-tricine polyacrylamide homogenous gel using the buffer system (10 x TG-SDS
READY PACK, Amersco, Olt, USA).
After transferring onto nitrocellulose
membranes, membranes were blocked in Tris-buffered saline (TBS) containing
0.3-1.0% nonfat milk (Bio-Rad Laboratories, Hercules, CA, USA) containing 0.1%
Tween 20 (Research Biochemicals, Inc., MA, USA), for 1 h at room temperature with
agitation and 4 h at 4°C with stationary.
The membrane was incubated with the
following primary antibodies; anti-signal tranducer and activator of transcription 3
(STAT3) (rabbit polyclonal, 1:1000, Cell signaling Technology, Inc., MA, USA),
anti-phosphorylated-STAT3 (rabbit polyclonal, 1:1000, Cell signaling Technology, Inc.,
MA, USA), anti-ionized calcium binding adaptor molecule 1 (Iba-1) (rabbit polyclonal
1:1000,
Wako
pure
Chemicals,
industrie,
Ltd,
Osaka,
Japan),
anti-phosphorylated-extracellular signal-regulated kinase (p-ERK) (mouse monoclonal,
1:500, Santa cruz biotechnology, inc., CA, USA), glyceraldehyde-3-phosphate
dehydrogenase (GAPDH) (mouse monoclonal, 1:400,000, Chemicon International, Inc.)
overnight at 4°C. The membrane was washed in TBS containing 0.05% Tween 20
(TTBS), followed by 2 h of incubation at room temperature with horseradish peroxidase
(HRP)-conjugated goat anti-rabbit IgG or HRP-conjugated anti-mouse IgG (Southern
Biotechnology Associates, Inc., Birmingham, AL, USA) diluted 1:10,000 in TBS
containing 0.3-1.0% nonfat milk containing 0.1% Tween 20. After this incubation, the
membranes were washed in TTBS. The antigen–antibody peroxidase complex was then
finally detected by enhanced chemiluminescence (Pierce, Rockford, IL, USA)
according to the manufacturer’s instructions and visualized by exposure to Amersham
Hyperfilm (Amersham Life Sciences, Arlington Heights, IL, USA).
RNA
preparation
and
semi-quantitative
analysis
by
reverse
transcription-polymerase chain reaction
Total RNA obtained from the mouse spinal cord was extracted using the SV Total
RNA Isolation system (Promega Co., Madison, WI, USA) as described previously 5.
After animals were decapitated, L3-5 lumbar dorsal root ganglia (DRGs) were rapidly
removed, and then the attached sciatic nerve and dorsal roots were transected adjacent
to the ganglion. Total RNA obtained from the mouse DRG was extracted using
ISOGENE (NIPPON GENE Co., Tokyo, Japan) according to the manufacturer’s
instructions. First-
of a
PCR solution containing 0.8 mM MgCl , dNTP mix, and DNA polymerase with
synthesized primers of MCP-3 (sense: 5'-TCT GTG CCT GCT GCT CAT AG-3',
antisense: 5'-CTT TGG AGT TGG GGT TTT CA-3'), MCP-1 (sense: 5'-AGC CAG
ATG CAG TTA ACG C-3', antisense: 5'-CTG ATC TCA TTT GGT TCC GA-3'),
RANTES (sense: 5’-GGT ACC ATG AAG ATC TCT GCA-3’, antisense: 5’-AAA CCC
TCT ATC CTA GCT CAT-3’), PU.1 (sense: 5’-CGG ATG TGC TTC CCT TAT CAA
AC-3’, antisense: 5’-TGA CTT TCT TCA CCT CGC CTG TC-3’), NF-IL-6 (sense:
5’-GGC CCT GAG TAA TCA CTT AAA GA-3’, antisense: 5’-ACA TAC GCC TCT
TTT CTC ATA G-3’), IL-6 (sense: 5'-ATG AAG TTC CTC TCT GCA AGA GAC T-3',
antisense: 5'-CAC TAG GTT TGC CGA GTA GAT CTC-3'), leukemia inhibitory factor
(LIF) (sense: 5'-GGC AAC CTC ATG AAC CAG ATC A-3', antisense: 5'-GCA AAG
CAC ATT GCT GAG GAG GC-3'), ciliary neurotrophic factor (CNTF) (sense: 5'-TGG
CTA GCA AGG AAG ATT CG-3', antisense: 5'-ATT GCC TGA TGG AAG TCA CC-3'),
cardiotrophin-1 (CT-1) (sense: 5'-CGG CCA ACA GCA CTG CAG GCA TC-3',
antisense: 5'-AAG CTC CCT GCA GAG AGG AGA GC-3'), oncostatin-M (OSM)
(sense: 5'-AAC ACT GCT CAG TTT GAC CC-3', antisense: 5'-ACA GTG CTC AGG
AAG TGA GG-3') or GAPDH (sense: 5’-CCC ACG GCA AGT TCA ACG G-3’,
antisense: 5’-CTT TCC AGA GGG GCC ATC CA-3’). Samples were heated to 94˚C for
3 min, 55˚C for 1 min, and 72˚C for 1 min, and then subjected to 29 cycles of 94˚C for
30 sec, 55˚C for 1 min, and 72˚C for 1 min. The final incubation was at 72˚C for 7 min.
The mixture was subjected to 2% agarose gel for electrophoresis with the indicated
markers and primers for the internal standard (GAPDH). Each sample was applied to
more than two lanes in the same gel. The agarose gel was stained with ethidium
bromide and photographed with UV transillumination.
The intensity of the bands was
analyzed and quantified by computer-assisted densitometry using the image-analysis
software ImageJ. For the control, the different intensities of each band from the sample
of sham-operated mice or sham-operated wild-type mice were analyzed, and the
average intensity was calculated.
Next, each control intensity was again compared
with the average intensity to calculate the standard error. Under these conditions, the
intensities of bands for samples obtained from nerve-ligated mice were analyzed and
compared with the average intensity for sham-operated mice.
Finally, the % of the
control with the standard error for each sample was quantified.
Quantitative analysis by Real-time reverse transcription-polymerase chain
reaction
A KAPA SYBR® FAST qPCR kit (Kapa Biosystems, Inc., MA, USA) was used as
the basis for the reaction mixture in the real-time PCR assay. Each gene prepared by
the above procedure was amplified in 20 l of a PCR solution containing 10 l of the
SYBR® FAST qPCR Master Mix with synthesized primers for MCP-3 (sense: 5'-TCT
GTG CCT GCT GCT CAT AG-3', antisense: 5'-CTT TGG AGT TGG GGT TTT CA-3')
or
-actin
(sense:
5’-CAGCTTCTTTGCAGCTCCTT-3’,
antisense:
5’-TCACCCACATAGGAGTCCTT-3’). In addition to each sample, each test run
included a no-target control that contained reaction mixture and PCR-grade water.
RT-PCR with a Mastercycler EP realplex (Eppendorf, Wesseling Berzdorf, Germany)
was performed with the following cycling conditions:
94˚C for 3 min, 55˚C for 1 min,
and 72˚C for 1 min, followed by cycled 29 cycles of 94˚C for 30 sec, 55˚C for 1 min,
and 72˚C for 1 min. The final incubation was at 72˚C for 7 min. Fluorescence detection
was conducted after each extension step.
Chromatin immunoprecipitation
A chromatin immunoprecipitation (ChIP) assay was performed as described
previously 6,7 with minor modifications. Briefly, the ipsilateral side of the mouse spinal
cord tissue was dissected as described above and cross-linked, and then tissue was lysed.
Fifteen μg of sonicated chromatin extracted from the mouse spinal cord was diluted in 1
mL ChIP dilution buffer (1.1%w/v DOC, 1.1%w/v Triton X-100, 167 mM NaCl, 50
mM Tris-HCl pH8.0) and then immunoprecipitated using 2 μg of specific antibodies
against acetylated histone H3 (Millipore), H3K4 trimethylation (Wako Pure Chemicals),
H3K9 trimethylation (Millipore), and H3K27 trimethylation (Millipore), overnight at
4ºC. The immuno-complex was collected by Dynabeads Protein A (Invitrogen Dynal
AS, Oslo, Norway), and DNA was recovered with RNaseA treatment, Proteinase K
treatment followed by isopropanol precipitation. Immunoprecipitated DNA was
dissolved in 50 l of 1 x TE and 1 l was used for quantitative PCR. Quantitative PCR
was performed as described previously8. The primer sequences were; MCP-3 (sense:
5’-CCT GTT TCT AGA CTC CAA GCT CC-3’, antisense 5’-ATT CCA ACC AGC
TCA GCT ACT AT-3’), MCP-1 (sense: 5’-ATT TGC TCC CAG GAG TGG C-3’,
antisense 5’-GGA GTC AGG CAG GGT GCT T-3’), RANTES (sense: 5’-CTG GAC
TGG AGG GCA GTT AG-3’, antisense 5’-TTG GGG AGT TTC CAC AAA AG-3’).
Immunohistochemistry using the mouse spinal cord-slice sections
One to 2 weeks after partial sciatic nerve ligation, mice were deeply anesthetized
with 3% isoflurane and intracardially perfusion-fixed with freshly prepared 4%
paraformaldehyde in 0.1 M in phosphate-buffered saline (PBS, pH 7.4).
After
perfusion, the sikn and viscera were removed. Then, the ribs were counted, and the most
caudal rib marked with ink.
The most craniad vertebra that lacked an articulation with
a rib at its rostral margin was regarded as the L1 vertebra, and was also marked.
Further dissection was performed to expose the vertebral and sacral bones in order to
identify the sites of fusion of the lower lumbar vertebrae. Then, L4 spinal cord was
quickly removed. The lumbar spinal cord sections were post-fixed in 4%
paraformaldehyde for 2 h, and permeated with 20 (w/v)% sucrose solution in 0.1 M
PBS for 24 h and 30 (w/v)% sucrose solution in 0.1 M PBS for 48 h at 4˚C with
agitation.
The lumbar spinal cord sections were then frozen in an embedding
compound (Sakura Finetechnical, Tokyo, Japan) on isopentane using liquid nitrogen
and stored at -30˚C until use. Frozen segments were cut with a freezing cryostat
(Leica CM 1510, Leica Microsystems AG, Wetzlar, Germany) at a thickness of 10 m
and thaw-mounted on poly-L-lysine-coated glass slides.
The sections were blocked in 7% normal horse serum (NHS) or 10% normal goat
serum (NGS) in 0.01 M PBS for 1 h at room temperature and then incubated for 48 h at
4˚C with the following primary antibodies: anti-MCP-3 (goat polyclonal, 1:200, R&D
systems, Inc., MN, USA), anti-neuron-specific nuclear protein NeuN (mouse
monoclonal, 1:250, Millipore Co., MA, USA), anti-glial fibrillary acidic protein
(GFAP) (rabbit polyclonal, 1:40, Nichirei Bioscience Co, Tokyo, Japan), anti-Iba-1
(rabbit
polyclonal,
1:200,
Wako
Pure
Chemical,
Industrie,
Ltd.,
Japan),
anti-metabotropic glutamate subtype 5 receptor (mGluR5) (rabbit polyclonal, 1:3000,
Upstate USA, Inc., VA, USA),
anti-protein kinase C (PKC) (rabbit polyclonal,
1:1000, Santa Cruz Biotechnology) and anti-microtubule associated protein-2 (MAP2)
(mouse monoclonal, 1:300, Chemicon International, Inc.).
The antibody was then
rinsed and incubated with an appropriate secondary antibody conjugated with Alexa
FluorTM 488 and/or 546 (Molecular Probes, Inc., Eugene, OR, USA) for 2 h at room
temperature.
The slides were then cover-slipped with Dako Huorescent mounting medium (Dako,
Denmark).
All sections were observed with a light microscope (Olympus BX-53;
Olympus) and photographed with a digital camera (CoolSNAP HQ; Olympus).
Digitized images of superficial laminae of the spinal dorsal horn sections were captured
at a resolution of 1316 x 1035 pixels with camera.
Preparation of spinal microglial cells
Purified spinal glial cells were grown as follows. The mouse spinal cord was
obtained from newborn ICR mice, minced, and treated with trypsin (0.025%, Invitrogen,
Carlsbad, CA) dissolved in PBS. After enzyme treatment at 37˚C for 15 min, cells were
dispersed by gentle agitation through a pipette and plated on a flask. Four weeks after
culture, floating microglia was collected from cultures by shaking flasks, then the cells
were seeded at a density of 5 x 104 cells/mL. The cells were maintained for 3 days in
Dulbecco's Modified Eagle Medium (DMEM) (Life Technologies Japan Ltd., Tokyo,
Japan) supplemented with 5% precolostrum newborn calf serum (Life Technologies
Japan), 5% heat-inactivated (56 ˚C, 30 min) horse serum (Life Technologies Japan), 10
U/ml penicillin and 10 g/ml streptomycin in a humidified atmosphere of 95% air and
5% CO2 at 37˚C.
These cells were treated with normal medium or recombinant
MCP-3 (10 ng/mL) with or without a CCR2 antagonist RS102895 (10 M) for 1 day,
and used as activated glial cells in the present study.
Immunohistochemistry using purified spinal microglia
Purified spinal microglia were identified by immunofluorescence using anti-Iba-1
(1:250, Wako Pure Chemicals) followed by incubation with an appropriate secondary
antibody conjugated with Alexa 488 (Molecular Probes). Stained cells were mounted
on glass slides and viewed using an Olympus BX-53 (Olympus). Each experimental
condition was repeated for three independent culture preparations.
Measurement of thermal hyperalgesia
To assess the sensitivity to thermal stimulation, each of the hind paws of mice was
tested individually using a thermal stimulus apparatus (model 33 Analgesia Meter; IITC
Inc./Life Science Instruments, Woodland Hills, CA, USA).
The intensity of the
thermal stimulus was adjusted to achieve an average baseline paw-withdrawal latency
of approximately 8 to 10 sec in naive mice. Only quick hind paw movements (with or
without licking of the hind paws) away from the stimulus were considered to be a
withdrawal response. Paw movements associated with locomotion or weight-shifting
were not counted as a response. The paws were measured alternating between the left
and right with an interval of more than 3 min between measurements.
The latency of
paw withdrawal after the thermal stimulus was determined as the average of 3 trials per
paw on each side. Before the behavioral responses to the thermal stimulus were tested,
mice were habituated for 1 h in an acrylic cylinder (15 cm high and 8 cm in diameter).
Under these conditions, the latency of paw withdrawal in response to a thermal stimulus
was tested. The latency of paw withdrawal in response to a thermal stimulus was
measured before and after partial sciatic nerve ligation. To investigate the effect of a
single intrathecal treatment with MCP-3 (0.1, 0.3, 1 pmol/mouse) and IL-6 (1
pmol/mouse) in normal mice or intrathecal treatment with anti-MCP-3 antibody, a
selective CCR2 antagonist, RS102895 and recombinant mouse gp130/Fc chimera
(gp130/Fc) in nerve-ligated mice on the thermal latency, the latency of paw withdrawal
was measured before and after injection until the latency returned to the baseline.
Measurement of tactile allodynia
To quantify the sensitivity to a tactile stimulus, paw withdrawal in response to a
tactile stimulus was measured using von Frey filaments (North Coast Medical, Inc.,
Morgan Hill, CA, USA) with a bending force of 0.02 g. The von Frey filament was
applied to the plantar surface of the hind paw for 3 sec, and this was repeated 3 times at
intervals of at least 5 sec. Each of the hind paws was tested individually.
Paw
withdrawal in response to a tactile stimulus was evaluated by scoring as follows: 0, no
response; 1, a slow and slight withdrawal response; 2, a slow and prolonged flexion
withdrawal response (sustained lifting of the paw) to the stimulus; 3, a quick withdrawal
response away from the stimulus without flinching or licking; 4, an intense withdrawal
response away from the stimulus with brisk flinching and/or licking. The latency of paw
withdrawal in response to a tactile stimulus was determined as the average of 2 trials per
paw on each side. Paw movements associated with locomotion or weight-shifting
were not counted as a response. The paws were measured alternating between left and
right with an interval of more than 3 min between measurements.
Before the
behavioral responses to a tactile stimulus were tested, mice were habituated for 1 h on
an elevated nylon mesh floor. Under these conditions, paw withdrawal in response to
a stimulus was measured before and after partial sciatic nerve ligation. To investigate
the effect of a single intrathecal treatment with MCP-3 (0.1, 0.3, 1 pmol/mouse) and
IL-6 (1 pmol/mouse) in normal mice or intrathecal treatment with anti-MCP-3 antibody,
a selective CCR2 antagonist, RS102895 and recombinant mouse gp130/Fc chimera
(gp130/Fc) in nerve-ligated mice on the tactile latency, the latency of paw withdrawal
was measured before and after injection until the latency returned to the baseline.
Functional imaging
Experiments were performed with a Unity Inova spectrometer (Varian, Palo Alto,
CA) which was interfaced to a 9.4-T/31-cm horizontal bore magnet equipped with
actively shielded gradients capable of 300 mT/m in a risetime of 500 sec (Magnex
Scientific, Abingdon, UK).
During the measurements, mice were slightly
anaesthetized with isoflurane (0.5 - 1%).
Mice were then transferred to a cradle
designed to fit inside the probe of the MR system. A continuous fMRI scanning
protocol was used to study changes in brain signal intensity using T2-weighted blood
oxygenation level-dependent (BOLD) contrast.
A functional series was acquired using
the Echo Planar Imaging Technique (EPI: matrix = 64 x 64, TR = 2000 ms, TE = 35 ms,
2 acquisitions, slice thickness = 1 mm, field of view = 25.6 x 25.6 mm2). Anatomical
scans with a high spatial resolution were collected using a fast spin echo pulse sequence
(matrix = 256 x 256, TR = 2000 ms, TE = 45 ms, slice thickness = 1mm, field of view =
25.6 x 25.6 mm2).
To investigate the effect of a single intrathecal treatment with MCP-3 (1
pmol/mouse), ICR mice were anesthetized at 3 h after intrathecal injection of MCP-3,
and heat stimulation was applied to the right hindpaw. Contact heat stimulation was
performed using a custom-made, computer-controlled Peltier heating and cooling
device. Peltier elements with a surface measuring 8.3 x 8.3 mm were fixed at the right
hindpaw. Starting at a baseline of 34°C, a stimulation temperature of 43-46°C was
reached after 18 sec at 0.67°C/sec.
The stimulation temperature plateau was held for
20 sec. Over the subsequent 22 sec, the temperature was dropped linearly back to the
baseline.
Data analysis was carried out using FEAT (http://www.fmrib.ox.ac.uk) software
packages.
Z (Gaussianised T/F) statistic images were thresholded using clusters
determined by Z>2.3 and a (corrected) cluster significance threshold of P=0.05.
Regions of interest (ROI) was manually selected and statistical analyses were performed
using an ImageJ. ROI were drawn according to an atlas of the mouse brain (Franklin
and Paxinos 1997). BOLD signal intensity values in each ROI were extracted and
normalized to the time of baseline (expressed as a percent change from baseline).
Drugs
The drugs used in the present study were recombinant mouse IL-6 (R&D Systems
Inc.), recombinant mouse MCP-3 (R&D Systems Inc.), the selective CCR2 antagonist
RS102895 (Sigma Chemical Co.), gp130/Fc (R&D Systems Inc.) and a functional
blocking antibody to MCP-3 (30 g/mL, R&D Systems Inc.). IL-6 (1 pmol/mouse),
MCP-3 (0.1, 0.3, 1.0 pmol/mouse) and gp130/Fc (10 ng/mouse) was dissolved in sterile
PBS containing 0.1% bovine serum albumin. RS102895 (100 ng/mouse) was dissolved
in saline containing 1% dimethylsulfoxide (DMSO).
Statistical analysis
The data are presented as the mean ± s.e.m.
The statistical significance of
differences between the groups was assessed by one-way analysis of variance
(ANOVA) (for pain-like behaviors) or Student’s t-test.
METHODS REFERENCES:
1.
Hylden, J.L. & Wilcox, G.L. Intrathecal morphine in mice: a new technique. Eur
J Pharmacol. 67, 313-316 (1980).
2.
Malmberg, A.B. & Basbaum, A.I. Partial sciatic nerve injury in the mouse as a
model of neuropathic pain: behavioral and neuroanatomical correlates. Pain. 76,
215-222 (1998).
3.
Narita, M., et al. Involvement of spinal protein kinase Cgamma in the
attenuation of opioid mu-receptor-mediated G-protein activation after chronic
intrathecal administration of [D-Ala2,N-MePhe4,Gly-Ol(5)]enkephalin. J
Neurosci. 21, 3715-3720 (2001).
4.
Bradford, M.M. A rapid and sensitive method for the quantitation of microgram
quantities of protein utilizing the principle of protein-dye binding. Anal Biochem.
72, 248-254 (1976).
5.
Narita, M., et al. Enhanced mu-opioid responses in the spinal cord of mice
lacking protein kinase Cgamma isoform. J Biol Chem. 276, 15409-15414
(2001).
6.
Takeshima, H., et al. The presence of RNA polymerase II, active or stalled,
predicts epigenetic fate of promoter CpG islands. Genome Res. 19, 1974-1982
(2009).
7.
Tsankova, N.M., Kumar, A. & Nestler, E.J. Histone modifications at gene
promoter regions in rat hippocampus after acute and chronic electroconvulsive
seizures. J Neurosci. 24, 5603-5610 (2004).
8.
Nakajima, T., et al. The presence of a methylation fingerprint of Helicobacter
pylori infection in human gastric mucosae. Int J Cancer. 124, 905-910 (2009).