Materials and Methods
Animal
Adult Sprague Dawley rats (MGI Cat# 5651135, RRID: MGI:5651135; sex:
male; weight: 250-300 g) (Samkako, Osan, Korea) were used in this study.
Animals were housed under controlled conditions (20-24°C, 55-65%
humidity, 12 h light/dark cycle), and have been given free access to
water and standard laboratory chow (5L79, PMI Nutrition International,
St Louis, MO). All animal experiments were performed in accordance with
the Guidelines and Policies for Rodent Survival Surgery provided by the
Animal Care Committee of the Kyung Hee University (Permission number:
KHUASP(SE)-17-059). Animal studies are reported in compliance with the
ARRIVE guidelines and with the recommendations made by the British
Journal of Pharmacology (Kilkenny et al.,
2010).
SCI surgery
SCI was performed using New York University (NYU) impactor in accordance
with previously described methods (Lee et
al., 2014a). Experimental induction of a contusive SCI in a rat model
using the NYU impactor device has been validated as an analog to human
SCI. A comparison between the rat model of SCI and human SCI shows
functional electrophysiological and morphological evidence of similar
patterns recorded in motor evoked potentials and somatosensory evoked
potentials as well as high-resolution magnetic resonance imaging
(Basso et al., 1995). In brief, rats were
anesthetized with chloral hydrate (500 mg∙kg-1) by
intraperitoneal (i.p.) injection and laminectomy was performed at the
T9-T10 level, exposing the spinal cord beneath without disrupting the
dura. The spinous processes of T8 and T11 were then clamped to stabilize
the spine, and the exposed dorsal surface of the cord was subjected to
moderate contusion injury (10 g X 25 mm) at the T9-T10 level using a NYU
impactor. Throughout the surgical procedure, body temperature was
maintained at 37 ± 0.5°C with a heating pad (Biomed S.L., Alicante,
Spain). After the injury, the muscles and skin were closed in layers,
and rats were placed in a temperature and humidity-controlled chamber
overnight. Postoperatively, rats were received subcutaneously
supplemental fluids (5 ml, lactated ringer) and antibiotics (gentamicin,
5 mg∙kg-1, intramuscular injection) once daily for 5
d. The bladder was emptied manually three times per day until reflexive
bladder emptying was established.
Drug treatment
Animals were randomly divided into three experimental groups (Sham,
Vehicle, and GA-treated group) (Fig. 1). GA (Cayman Chemical, Ann Arbor,
MI) was dissolved in 0.9% saline and rats were given GA (50
mg∙kg-1) immediately after SCI by i.p. injection and
then received the same dose of GA at 6 h and 12 h, and then further
treated once a day for 7 d. Based on our preliminary study, we found
that a dose of 50 mg/kg of GA was an optimal dose for the reduction of
BSCB disruption after SCI (Fig. 4D) and thus we used 50 mg/kg of GA
throughout this study. Sham-operated animals received no pharmacological
treatment and vehicle group received equivolumetric administration of
0.9% saline.
Tissue preparation
At indicated time points, rats were anesthetized with chloral hydrate
(500 mg∙kg-1, i.p.) and perfused via cardiac puncture
initially with 0.1 M PBS and subsequently with 4% paraformaldehyde in
0.1 M PBS. A 20-mm spinal cord, centered at the lesion site, was
dissected out, post-fixed by immersion in the same fixative (4%
paraformaldehyde) for 5 h and placed in 30% sucrose in 0.1 M PBS. The
segment was embedded in OCT for frozen sections, and longitudinal or
transverse sections were then cut at 10 or 20 µm on a cryostat (CM1850;
Leica, Wetzlar, Germany). For molecular work, rats were perfused with
0.1 M PBS and the segments of the spinal cord (10 mm) including the
lesion site were isolated and frozen at -80°C until use.
Molecular docking study
The structure used as a template for the structure-based docking study
was prepared by using the X-ray structure (PDB code 4ASK)
(Kruidenier et al., 2012). All the water
molecules and ligand were removed, and the hydrogen atoms were added.
The coordinate file for the structure of inhibitor compounds was
constructed in Sybyl X-2.1.1 and energetically minimized using a Tripos
force field with Gasteiger-Huckel charges. Docking was carried using
Surflex-Dock GeomX module interfaced in Sybyl X-2.1.1 by generating
protocol using the co-crystallized ligand in the binding site
(Jain, 2003). The ligand was docked to
the receptor and analyzed with the original ligand bound to the receptor
to validate the method. The generated compounds were also docked into
the binding site and the results were analyzed and ranked by Total Score
(-logKd).
In vitro histone methylation assay
In vitro Jmjd3 inhibition assay was modified from Epigenase
Jmjd3/UTX demethylase activity/Inhibition assay kit (P-3084, Epigentek,
Farmingdale, NY). As briefly described, acid-extracted histones were
incubated with a Jmjd3 enzyme (E24026-1, Epigentek) in an assay buffer
(0.05% Tween-20, 0.05% BSA, 10 μM
NH4FeSO4, 200 μM ascorbic acid, 2 μM
α-ketoglutaric acid, 25 mM HEPES, pH 7.5) with each inhibitor as a
proper concentration for 2 h at 37℃. The demethylation reaction was
stopped by adding a 5X SDS sample buffer and boiling for 5 min. Western
blotting was carried out using anti-H3K27me3 (Millipore, Cat# 07-449,
RRID: AB_310624).
Endothelial cell culture and oxygen-glucose deprivation
(OGD)/reperfusion
A mouse brain endothelial cell line, bEnd.3 (ATCC, Cat# CRL-2299, RRID:
CVCL_0170) was cultured as previously described
(Lee et al., 2012b;
Lee et al., 2012c). Prior to each
experiment, cells were seeded onto 6-well
(5 × 105 cells/well) plates. To achieve OGD, cells
were transferred to a humidified anaerobic chamber (APM-30D, Astec,
Fukuoka, Japan) under an atmosphere of 0.1% O2, 5%
CO2 balanced with 95% N2. The culture
medium was replaced three times with deoxygenated and glucose-free DMEM.
The cells were treated with GA (10 μM) for 30 min before OGD. At the end
of the OGD period, cells were placed under normoxic conditions and the
media was quickly replaced with 25 mM glucose containing DMEM. Control
cells were cultured in DMEM with 25 mM glucose under normoxia. GA (10
μM) was dissolved in 0.9% saline, which had no effect on cell
viability.
Chromatin immunoprecipitation (ChIP)
ChIP assay was performed as previously described
(Lee et al., 2016;
Lee et al., 2012c). The primary
antibodies used in ChIP assay are as follows: Jmjd3 (Abgent, Cat#
AP1022a, RRID: AB_889281), H3k27me3 (Millipore), normal IgG (Santa Cruz
Biotechnology, Cat# sc-2027, RRID: AB_737197). Cells were crosslinked
using 1% formaldehyde in PBS for 15 min at room temperature. To stop
the cross-linking, 1.25 M glycine was added (1% final concentration).
After washing with ice-cold PBS, the cells in SDS lysis buffer (1% SDS,
10 mM EDTA, 50 mM Tris-HCl, pH 8.1) was sonicated until the DNA
fragments were 300-500 bp in size. The extracts were subsequently
centrifuged, and the resulting soluble chromatin solutions were diluted
10 fold with ChIP dilution buffer (0.01% SDS, 1.1% Triton X-100, 1.2
mM EDTA, 167 mM NaCl, 16.7 mM Tris-HCl, pH 8.1). The specific antibodies
or IgG were added into soluble chromatin solution for overnight at 4 °C
and followed by protein A-sepharose beads (Sigma-Aldrich, St. Louis, MO)
for 2 h. The beads were extensively washed with low salt wash buffer
(0.1% SDS, 1% Triton X-100, 2 mM EDTA, 150 mM NaCl, 20 mM Tris-HCl,
pH8.1), high salt wash buffer (0.1% SDS, 1% Triton X-100, 2 mM EDTA,
500 mM NaCl, 20 mM Tris-HCl, pH 8.1), LiCl wash buffer (0.25 M LiCl, 1%
NP-40, 1% deoxycholate, 1 mM EDTA, 10 mM Tris-HCl, pH 8.1), and finally
with TE buffer. After elution of the DNA/protein with 1% SDS,
crosslinking was reversed for 6 h at 65 °C. The DNA was recovered using
a QIAquick spin column (Qiagen, Valencia, CA). Real time-PCR was
performed with a Stratagene Mx3000P using primers that cover mouse,Mmp-3 (5′-TTC CGC CTT TTT TGT TCA-3′ and 5′-CCA CTC AAA AAC AGG
TCT ATA ATT T-3′), and Mmp-9 (5′-CCC AGG CTC ATC TTT CCT TCC
CC-3′ and 5′-CCC ATC CCC ACA CTG TAG GTT C-3′). The relative proportions
of immunoprecipitated fragments were determined using the ΔCt
comparative method based on the threshold cycle (Ct) value for each PCR
reaction and normalized to input genomic DNA.
Evans blue assay
BSCB permeability was investigated with Evans Blue dye extravasation as
previously described (Lee et al., 2012a).
In brief, 5 ml of 2% Evans blue dye (Sigma) was administered via i.p.
injection at 24 h after SCI and then perfused with 0.1M PBS at 3 h
later. The spinal cords (5 mm) including lesion epicenter were
homogenized and the fluorescence intensity was measured on a Gemini™ XPS
and EM Microplate Readers (Molecular device, Sunnyvale, CA) at 620 nm
excitation/680 nm emission. The Dye in samples was determined as
micrograms per gram of tissue from a standard curve plotted using known
amounts of dye.
Immunohistochemistry
Frozen sections were processed for immunohistochemistry with antibodies
against myeloperoxidase (MPO; 1:100; Agilent, Cat# A0398, RRID:
AB_2335676), ED-1 (1:1,000; Bio-Rad, Cat# MCA341R, RRID: AB_2291300),
cleaved caspase-3 (1:100; Cell Signaling Technology, Cat# 9661, RRID:
AB_2341188), CC1 (1:100; Abcam,, Cat# ab16794, RRID: AB_443473),
Jmjd3 (1:100; Abcam, Cat# ab38113, RRID: AB_943898) and RECA1 (1:100;
Bio-Rad, Cat# MCA970GA, RRID: AB_567193) as previously described
(Lee et al., 2014b). For quantification
of MPO or ED-1 intensity, serial transverse sections (20 μm thickness)
were collected every 100 μm section rostral and caudal 3,000 μm to the
lesion site (total 60 sections). Digital images of MPO- or ED-1-stained
tissues were obtained and quantified the entire fluorescent intensity of
each transverse section above the threshold by using MetaMorph software
(Molecular devices) and averaged. The threshold value was at least three
times the background and the backgrounds were quantified and normalized
to the primary antibody omitted control. Immunostaining control studies
were performed by omission of the primary antibodies, by replacement
primary antibodies with non-immune, control antibody, and by
pre-absorption with an excess (10 µg/ml) of the respective antigens. For
double labeling, fluorescein isothiocyanate - or cyanin 3-conjugated
secondary antibodies (Jackson ImmunoResearch Labs, Cat# 111-165-003,
RRID: AB_2338000) was used. Also, nuclei were labeled with DAPI
according to the protocol of the manufacturer (Thermo Fisher Scientific,
Cat# D3571, RRID: AB_2307445). In all controls, reaction to the
substrate was absent if the primary antibody was omitted or if the
primary antibody was replaced by a nonimmune control antibody. For
quantification of cleaved caspase-3-positive oligodendrocytes (cleaved
caspase-3/CC1 double positive), serial transverse sections (10 μm
thickness) were collected every 200 μm from 4,000 μm rostral to 4,000 μm
caudal to the lesion site (total 40 sections). Cleaved
caspase-3-positive oligodendrocytes in the white matter (WM) in each
section were counted and averaged. Serial sections were also stained
with Cresyl violet acetate for histological analysis.
Cell counting of viable ventral motor neurons (VMNs)
One day after injury, the number of VMNs was counted and assessed. The
criteria for VMN counting was based on the previous report
(Lee et al., 2014b). In brief, we
determined the cells located in the lower ventral horn and were larger
than half of the sampling square (20 x 20 μm) as a VMN. The cells above
the line at 150 μm ventral from the central canal were excluded. For
counting of the total number of VMN, serial transverse sections (20 µm
thickness) were collected every millimeter section rostral and caudal 8
mm to the lesion site and stained with Cresyl violet acetate. Motor
neurons were manually counted from each field and analyzed by MetaMorph
software (Molecular Devices).
TUNEL staining
One and five days after injury, serial spinal cord sections (20 μm
thickness) were collected every 200 μm and processed for TUNEL staining
using an ApopTag in situ kit (Millipore, Cat# S7100, RRID:
AB_2661855), according to the manufacturer’s instructions. A DAB
substrate kit (Vector Laboratories, Burlingame, CA) was used for
peroxidase staining. Control sections were treated similarly, but
incubated in the absence of TDT enzyme, dUTP-digoxigenin, or
anti-digoxigenin Ab, and positive control sections were incubated in
DNase 1. TUNEL-positive cells in the gray matter (GM) at 1 d (total 40
sections) and in the WM at 5 d (total 100 sections) after SCI were
counted and quantified using a 20 x objective. Only those cells showing
morphological features of nuclear condensation and/or
compartmentalization in the GM and WM were counted as a TUNEL-positive
cell.
RNA isolation and RT-PCR
Total RNA at the indicated time points was isolated from spinal cord
segments (10 mm), centered at the lesion site by using TRIZOL reagent
(Invitrogen) and RT-PCR were performed as previously described
(Lee et al., 2012b;
Lee et al., 2003;
Yune et al., 2007). The primers used for
RT-PCR were synthesized by Genotech (Daejeon, Korea), and the sequences
of all primers are presented in Table 1. After amplification, PCR
products were subjected to a 1.5 or 3 % agarose gel electrophoresis and
visualized by ethidium bromide staining. The relative density of bands
(relative to sham value) was analyzed by the AlphaImager software (Alpha
Innotech Corporation, San Leandro, CA). The expression of GAPDH was used
as an internal control. Experiments were repeated three times and the
values obtained for the relative intensity were subjected to statistical
analysis. The gels shown in figures are representative of results from
three separate experiments.
Western blot
Total protein preparation at the indicated time points and Western blot
analysis were performed as previously described
(Lee et al., 2012a). Protein sample (30
µg) was separated on SDS-PAGE gel electrophoresis and transferred to
nitrocellulose membrane (Millipore). The membranes were blocked in 5%
non-fat skim milk or 5% bovine serum albumin in Tris-buffered saline
containing tween-20 (0.1%), and subsequently incubated with antibodies.
The primary antibodies used in Western blot are as follows. Jmjd3
(1:1,000; Abcam), H3K27me3 (1:1,000; Abcam, Cat# ab6002,
RRID:AB_305237), Histon H3 (1:1,000; Cell Signaling Technology, Cat#
9715, RRID: AB_331563), ED-1 (1:1,000; Bio-Rad), iNOS (1:1,000; BD
Biosciences, Cat# 610333, RRID: AB_397723), COX-2 (1:1,000; Cayman
Chemical, Cat# 160107, RRID: AB_10078833), cleaved caspase-3 (1:1,000;
Cell Signaling), ZO-1 (1:1,000; Thermo Fisher Scientific, Cat# 40-2200,
RRID: AB_2533456), and occludin (1:1,000; Thermo Fisher Scientific,
Cat# 40-4700, RRID: AB_2533468). β-tubulin (1:30,000; Sigma-Aldrich,
Cat# T4026, RRID: AB_477577) was used as a loading control. The
primary antibody was detected with a horseradish peroxidase-conjugated
secondary antibody (Jackson ImmunoResearch). Immunoreactive bands were
visualized by chemiluminescence using Supersignal (Thermo Scientific,
Rockford, IL). The densitometric values of the bands on Western blots
were obtained by AlphaImager software (Alpha Innotech Corporation) and
subjected to statistical analysis. Background in films was subtracted
from the optical density measurements.
Gelatin zymography
The activities of MMP-2 and MMP-9 by gelatin zymography were performed
using total protein (50 µg) at 1 d after injury as previously described
(Lee et al., 2012b). Total protein was
loaded on a Novex 10% zymogram gel (EC61752; Invitrogen) and separated
by electrophoresis. The gel was then incubated with renaturing buffer
(2.5% Triton X-100) at room temperature for 30 min to restore the
gelatinolytic activity of the proteins. After incubation with developing
buffer (50 mM Tris-HCl, pH 8.5, 0.2 M NaCl, 5 mM CaCl2,
0.02% Brii35) at 37°C for 24 h, the gel was stained with 0.5%
Coomassie and then destained with 40% methanol containing 10% acetic
acid until appropriate color contrast was achieved. The clear bands on
the zymogram were indicative of gelatinase activity. Quantification of
bands was performed by AlphaImager software (Alpha Innotech
Corporation). Experiments were repeated three times and the values
obtained for the relative intensity were subjected to statistical
analysis.
Behavioral tests
The examination of functional deficits after injury was conducted as
previously described (Basso et al., 1995;
Lee et al., 2003;
Rivlin & Tator, 1977;
Yune et al., 2007). Behavioral analyses
were performed by trained investigators who were blind as to the
experimental conditions. Open-field locomotion to test hindlimb
locomotor function was evaluated by using the Basso-Beattie-Bresnahan
(BBB) locomotion scale. BBB is a 22-point scale (scores 0-21) that
systematically and logically follows recovery of hindlimb function from
a score of 0, indicative of no observed hindlimb movements, to a score
of 21, representative of a normal ambulating rodent. For the inclined
plane test, rats were tested in two positions (right side or left side
up) on the testing apparatus. The maximum angle at which a rat could
maintain its position for 5 s without falling was recorded for each
position and averaged to obtain a single score for each animal. The
ability to control and place the hindlimb precisely was tested on a
horizontal grid and the analysis was performed by counting the number of
footfall (mistake) in foot placing. For the footprint analysis, both
forepaws and hind paws were dipped in red and blue dye (nontoxic) and
then walked across a narrow box (1 m long and 7 cm wide). The footprints
were then scanned, and digitized images were analyzed.
Axon counting and myelin staining
After behavioral tests, rats treated with vehicle and GA were perfused
at 35 d after injury, and frozen sections were prepared as described
above. For quantitative analysis of axonal density, serial coronal
sections collected every millimeter rostral and caudal 5 mm to the
lesion site were stained with an antibody specific for 200 kDa
neurofilament protein (NF200, 1:4,000; Sigma-Aldrich, Cat# N4142, RRID:
AB_477272). Some sections were processed for 5-hydroxytryptamine (5-HT,
1:5,000; ImmunoStar, Cat# 20080, RRID: AB_572263 N) staining. The ABC
method was used to detect labeled cells using a Vectastain kit (Vector
Laboratories). Axonal densities were determined within preselected
fields (40 × 40 μm, 1,600 μm2) at specific sites
within the ventral and dorsolateral funiculi as previously described
(Yune et al., 2007). Axons were manually
counted from each field and analyzed by MetaMorph software (Molecular
Devices). For myelin staining, selected slides were incubated in 0.1%
Luxol fast blue (Solvent Blue 38; Sigma) in acidified 95% ethanol
overnight at 60°C. Differentiation was performed with 0.05% lithium
carbonate as previous described (Lee et
al., 2018). Digital images of Luxol fast blue-stained tissues were
obtained by MetaMorph software (Molecular Devices).
Assessment of lesion volume
The measurement of lesion volume using rats tested for behavioral
analyses was performed as previously described
(Yune et al., 2008). Serial longitudinal
sections (10 μm) through the dorsoventral axis of the spinal cord were
used to measure the lesion volume. Every 50 μm section was stained with
Cresyl violet acetate. The lesion volume was determined by measuring the
area of cavitation at the injury epicenter using a low-power (1.25 x)
objective and then calculated by mean of a MetaMorph software (Molecular
Devices). Areas of each longitudinal level are determined, and the total
lesion volume was induced by numerical integration of sequential areas.
Statistical analysis
The biochemical assays and analysis of the results was carried out
without knowledge of the treatment groups (blinded). Each experiment
involved at least 5 independent samples (equal size) per randomized
group, and that statistical analysis was done using these independent
values. The statistical analysis was undertaken only for studies where
each group size was at least n = 5. The control and test values in
Western blot and RT-PCR were normalized to an internal standard (such as
β-Tubulin and GAPDH) to reduce variance. Western blot and RT-PCR were
normalized to the mean value of the experimental control group to set
Y-axis so the control group value was 1. The units for these normalized
data in the Y-axis were the fold of the control group’s mean value. All
statistical analyses were performed by SPSS 15.0 (SPSS Science, Chicago,
IL). Western blot and RT-PCR presented as the mean ± SD values and Evans
blue, BBB, grid walk and inclined plane test data are presented as the
mean ± SEM. Comparisons between vehicle and GA-treated groups were made
by unpaired Student’s t test. Multiple comparisons between groups were
performed one-way ANOVA. Behavioral scores from BBB and inclined plane
test were analyzed by repeated measures ANOVA (time vs treatment).
Tukey’s multiple comparison was used as Post hoc analysis. For each
parameter of the data presented, * indicates p < 0.05.