p53- and Bax-mediated apoptosis in injured rat spinal cord

Abstract

Spinal cord injury (SCI) induces a series of endogenous biochemical changes that lead to secondary degeneration, including apoptosis. p53-mediated mitochondrial apoptosis is likely to be an important mechanism of cell death in spinal cord injury. However, the signaling cascades that are activated before DNA fragmentation have not yet been determined. DNA damage-induced, p53-activated neuronal cell death has already been identified in several neurodegenerative diseases. To determine DNA damage-induced, p53-mediated apoptosis in spinal cord injury, we performed RT-PCR microarray and analyzed 84 DNA damaging and apoptotic genes. Genes involved in DNA damage and apoptosis were upregulated whereas anti-apoptotic genes were downregulated in injured spinal cords. Western blot analysis showed the upregulation of DNA damage-inducing protein such as ATM, cell cycle checkpoint kinases, 8-hydroxy-2'-deoxyguanosine (8-OHdG), BRCA2 and H2AX in injured spinal cord tissues. Detection of phospho-H2AX in the nucleus and release of 8-OHdG in cytosol were demonstrated by immunohistochemistry. Expression of p53 was observed in the neurons, oligodendrocytes and astrocytes after spinal cord injury. Upregulation of phospho-p53, Bax and downregulation of Bcl2 were detected after spinal cord injury. Sub-cellular distribution of Bax and cytochrome c indicated mitochondrial-mediated apoptosis taking place after spinal cord injury. In addition, we carried out immunohistochemical analysis to confirm Bax translocation into the mitochondria and activated p53 at Ser(3)(9)(2). Expression of APAF1, caspase 9 and caspase 3 activities confirmed the intrinsic apoptotic pathway after SCI. Activated p53 and Bax mitochondrial translocation were detected in injured spinal neurons. Taken together, the in vitro data strengthened the in vivo observations of DNA damage-induced p53-mediated mitochondrial apoptosis in the injured spinal cord.

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ORIGINAL PAPER
p53- and Bax-Mediated Apoptosis in Injured Rat Spinal Cord
Ramaprasada Rao Kotipatruni•Venkata Ramesh Dasari•
Krishna Kumar Veeravalli•Dzung H. Dinh•
Daniel Fassett•Jasti S. Rao
Accepted: 7 June 2011
? Springer Science+Business Media, LLC 2011
Abstract
endogenous biochemical changes that lead to secondary
degeneration, including apoptosis. p53-mediated mito-
chondrial apoptosis is likely to be an important mechanism
of cell death in spinal cord injury. However, the signaling
cascades that are activated before DNA fragmentation have
not yet been determined. DNA damage-induced, p53-acti-
vated neuronal cell death has already been identified in
several neurodegenerative diseases. To determine DNA
damage-induced, p53-mediated apoptosis in spinal cord
injury, we performed RT-PCR microarray and analyzed 84
DNA damaging and apoptotic genes. Genes involved in
DNA damage and apoptosis were upregulated whereas
anti-apoptotic genes were downregulated in injured spinal
cords. Western blot analysis showed the upregulation of
DNA damage-inducing protein such as ATM, cell cycle
checkpoint kinases, 8-hydroxy-20-deoxyguanosine (8-OHdG),
BRCA2 and H2AX in injured spinal cord tissues. Detection
of phospho-H2AX in the nucleus and release of 8-OHdG
Spinal cord injury (SCI) induces a series of
in cytosol were demonstrated by immunohistochemistry.
Expression of p53 was observed in the neurons, oligo-
dendrocytes and astrocytes after spinal cord injury.
Upregulation of phospho-p53, Bax and downregulation of
Bcl2 were detected after spinal cord injury. Sub-cellular
distribution of Bax and cytochrome c indicated mitochon-
drial-mediated apoptosis taking place after spinal cord
injury. In addition, we carried out immunohistochemical
analysis to confirm Bax translocation into the mitochondria
and activated p53 at Ser392. Expression of APAF1, caspase
9 and caspase 3 activities confirmed the intrinsic apoptotic
pathway after SCI. Activated p53 and Bax mitochondrial
translocation were detected in injured spinal neurons.
Taken together, the in vitro data strengthened the in vivo
observations ofDNA damage-induced
mitochondrial apoptosis in the injured spinal cord.
p53-mediated
Keywords
Microarrays ? p53 ? Spinal cord injury
Apoptosis ? Bax ? DNA damage ?
Abbreviations
ALS
ANOVA
APAF1
ATM
ATR
BRCA2
BSA
CHAPS
Amyotrophic lateral sclerosis
Analysis of variance
Apoptotic protease activating factor 1
Ataxia telangiectasia mutated
Ataxia telangiectasia and Rad3 related
Breast cancer 2, early onset
Bovine serum albumin
3-[(3-Cholamidopropyl)dimethylammonio]
propanesulfonic acid
Checkpoint homolog (S. pombe)
Central nervous system
Diaminobenzidine
40,6-Diamidino-2-phenylindole
dihydrochloride
CHK2
CNS
DAB
DAPI
Ramaprasada Rao Kotipatruni and Venkata Ramesh Dasari
contributed equally.
Electronic supplementary material
article (doi:10.1007/s11064-011-0530-2) contains supplementary
material, which is available to authorized users.
The online version of this
R. R. Kotipatruni ? V. R. Dasari ? K. K. Veeravalli ?
J. S. Rao (&)
Department of Cancer Biology and Pharmacology, University
of Illinois College of Medicine at Peoria, One Illini Drive,
Peoria, IL 61656, USA
e-mail: jsrao@uic.edu
D. H. Dinh ? D. Fassett ? J. S. Rao
Department of Neurosurgery, University of Illinois College
of Medicine at Peoria, Peoria, IL, USA
123
Neurochem Res
DOI 10.1007/s11064-011-0530-2

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DNA
DSB
DTT
EDTA
ELISA
FBS
FITC
GFAP
H2AX
HEPES
Deoxyribonucleic acid
Double-strand breaks
Dithiothreitol
Ethylenediaminetetraacetic acid
Enzyme-linked immunosorbent assay
Fetal bovine serum
Fluorescein isothiocyanate
Glial fibrillary acidic protein
Histone H2A.X
4-(2-hydroxyethyl)-1-piperazineethanesulfonic
acid
Horseradish peroxidase
Human umbilical cord blood derived stem cells
Oligodendrocyte transcription factor 2
Poly acrylamide gel electrophoresis
Phosphate buffered saline
Reverse Transcription based Polymerase chain
reaction
Phenyl methane sulfonyl fluoride
Spinal cord injury
Sodium dodecyl sulfate
Single-strand breaks
Staurosporine
Tris-acetate-EDTA
Terminal deoxynucleotidyl transferase dUTP
nick end labeling
HRP
hUCB
Olig2
PAGE
PBS
RT-PCR
PMSF
SCI
SDS
SSB
STP
TAE
TUNEL
Introduction
Neuronal apoptosis is mediated by the mitochondrial-
mediated pathway in a majority of neurodegenerative dis-
eases. For instance, in cerebral ischemia, neurodegenera-
tion is mediated by p53 activation and Bax translocation [1,
2]. Spinal cord injury (SCI) results in the initial physical
disruption of structures in the spinal cord (primary injury)
and in the generation of secondary events that collectively
injure the otherwise intact neighboring tissue. Secondary
pathogenesis of spinal cord injury is the most devastating
event caused by neuronal cell apoptosis. Our recent study
demonstrated the upregulation of mitochondrial apoptotic
genes such as Bad, Bid, Bid3, Bcl10, Bcl2a1, Bik and Bak1
in the injured rat spinal cords [3]. SCI induced by trauma
compresses and shears neuronal and glial cell types and
their processes in the immediate vicinity of the impact. As
a consequence, cells spill their intracellular contents into
the extracellular matrix. This pathophysiological occur-
rence initiates a cascade of biochemical changes that
results in necrotic and apoptotic cell death [4].
Although DNA damage may ensue from excitotoxicity
and other stresses possibly as a result of increased produc-
tion of reactive oxygen species, DNA damage is a common
activator of neuronal injury [5]. These cellular stresses are
linked to the activation of numerous downstream signaling
pathways such as the cyclin-dependent and stress-activated
kinases, DNA-dependent protein kinase, ATM and p53.
Once activated in neurons, p53 appears to have a major
impact on mitochondrial integrity, which revolves around
members of the Bcl-2 family [5]. p53 influences the
expression and/or activity of the pro-apoptotic proteins, Bax
and APAF1. Several of these proteins affect mitochondrial
membrane permeability (e.g., Bax) or the activation of
apoptotic mediators downstream of the mitochondria (e.g.,
APAF1). p53 promotes Bax translocation from the cytosol
to the outer membrane of the mitochondria [6]. The rela-
tionship between p53 expression and neuronal cell death
has been evaluated in numerous models of injury and dis-
ease [7]. Recently transcription factor p53 was identified in
degenerating the spinal cord of ALS patients [8]. p53 pro-
motes apoptosis by modulating the expression of select
target genes. Numerous pro-apoptotic genes are susceptible
to regulation by p53, including Bax. [9]. Bax was reported
to be a cytosolic protein in healthy living cells and upon
induction of apoptosis, it translocates to the mitochondria
[10–12]. This translocation process is rapid and occurs at an
early stage of apoptosis [12]. It has been reported that the
intracellular movement of Bax to the mitochondria depo-
larizes the mitochondria and induces the release of cyto-
chrome c, which causes the adaptor APAF1 to activate
caspases 9 and 3 and ultimately cellular demise [13, 14].
Finally, no studies have been reported to ascertain the role
of Bax in the secondary pathogenesis of spinal cord injury-
mediated neuronal cell death. Thus, our overall goal was to
determine the molecular mechanism(s) by which SCI elicits
its apoptotic effects on neuronal cells adjacent to the injured
spinal cord.
The present study is designed to demonstrate p53 acti-
vation and Bax-mediated mitochondrial apoptosis in injured
spinal cords of rat in vivo and spinal neurons in vitro. To
date, no information exists regarding the DNA damage
linked to neuronal apoptosis after spinal cord injury.
Therefore,this study wasdesignedtoinvestigate the level of
DNA damage and expression of genes involved in apoptosis
after SCI.
Experimental Procedures
Preparation of Primary Cell Cultures of Spinal Neurons
Primary cultures of spinal neurons were prepared from
embryonic 18-day Sprague–Dawley rats as previously
described [15]. Spinal cords were obtained from Brain Bits
(Springfield, IL) and triturated gently (8–10 times) in cold
Hibernate-E medium plus 19 B27-supplement (Invitrogen,
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Carlsbad, CA) and 1% penicillin-streptomycin (Invitrogen).
Once in suspension, the cells were diluted in 1 mL of the
samemediumandthenumberofviablecellswasdetermined
by Trypan blue exclusion. Cells were plated at a density of
0.25 9 106cells/lL on poly-D-lysine-coated (50 lg/mL)
plates in neurobasal medium. Cultures were maintained at
37?C in a humidified 5% CO2atmosphere, and all experi-
ments were performed after 12–15 days in culture. Cultures
were fed every 3 days with fresh medium. All experiments
were performed in neurobasal medium containing 2% anti-
oxidant-free B27-supplement. Under these culture condi-
tions, only neuronal cells survive. These cultures were
comprised of approximately 90–95% neuronal cells as
confirmed by immunocytochemical staining performed
accordingtothemanufacturer’sinstructionswithanti-NeuN
(Abcam, Cambridge, MA) or anti-neurofilament 200kDa
(Chemicon, Temecula, CA) antibodies.
Spinal Cord Injury of Rat
Moderate spinal cord injury was induced using the weight
dropdevice(NYUImpactor)asreportedpreviously[16,17].
Briefly, adult male rats (Lewis; 250–300 g) were anesthe-
tized with ketamine (100 mg/kg; ip) and xylazine (5 mg/kg;
ip) (Med-Vet International, Mettawa, IL). A laminectomy
was performed at the T9–T11 level exposing the cord
beneath without disrupting the dura, and the exposed dorsal
surface of the cord at T10 was subjected to a weight drop
impact using a 10 g rod (2.5 mm in diameter) dropped at a
height of 12.5 mm. The animals were sacrificed at different
time points (1, 3, 7, 14 and 21 days). The Institutional
Animal Care and Use Committee of the University Of Illi-
nois College Of Medicine at Peoria approved all surgical
interventions and post-operative animal care.
Determination of Caspase 9 Activity
A quantitative enzymatic activity assay was performed
using Caspase 9 Assay kit (R&D Systems, Minneapolis,
MN). Tissue lysates at different time points were incubated
at 37?C in buffer supplemented with substrate LEHD-7-
amino-4-trifluoromethyl coumarin (AFC) (25 lM) for
caspase 9. Protease activity of caspase 9 was monitored
using a substrate with an optimized bifunctional cell lysis/
activity buffer. Samples were assayed at 499 nm absor-
bance on the BioRad plate reader. Specific inhibition of
caspase 9 was achieved by the addition of caspase inhibitor
AC-LEHD-CHO.
Determination of Caspase 3 Activity
Caspase 3 activity of tissue lysates at different time points
was measured using a caspase 3 assay kit (Sigma, St. Louis,
MO). Injured and sham operated spinal cord tissues were
homogenized in 19 lysis buffer (50 mM HEPES, pH 7.4,
5 mM CHAPS, 5 mM DTT) and the homogenates were
then centrifuged at 20,0009g for 15 min at 4?C in a micro-
centrifuge. We analyzed the supernatants for caspase 3
activitylevelsusingthe96-wellplatemicroassaymethod.In
a final volume of 100 lL, supernatants (50 lg protein) of
each test sample were incubated for 30 min at room tem-
perature in the working solution containing Ac-DEVD-
AMC, a synthetic caspase 3 substrate. Absorbance was read
at 405 nm on an ELISA plate reader for a period of 30 min.
We used Ac-DEVD-CHO inhibitor for the inhibitor studies.
Caspase 3 activity was calculated using a p-nitro aniline
calibration curve. The data were plotted as A405versus time
for each sample and activity was calculated as pmol/min.
Staurosporine-Induced Apoptosis in Cultured Rat
Spinal Neurons
After 12–15 DIV (days in vitro), primary spinal neurons
(E18) were exposed to 2 lM staurosporine for 4 h at 37?C
to induce apoptosis. After the neurons had been exposed to
staurosporine for the required time, the cells were detached
from the plates by gentle scraping and were centrifuged at
5009g for 3 min. The supernatant was removed, and the
pellet was resuspended in PBS and centrifuged again at
5009g for 3 min to wash the cells. The supernatant was
discarded, and the cell pellet resuspended in 1 mL of
propidium iodide (50 lg/mL) solution. The samples were
analyzed using a flow cytometer (BD FACS Calibur, BD
Biosciences, Franklin Lakes, NJ).
Immunoblot Analysis
For immunoblot analysis, rats were euthanized, and
1–4 mm lengths of spinal cord from the injury site were
rapidly removed, weighed, and frozen at -80?C until
necessary for further experimentation. The tissues were
resuspended in 0.2 mL of homogenization buffer (pH 7.4,
250 mM sucrose, 10 mM HEPES, 10 mM Tris–HCl,
10 mM KCl, 1% NP-40, 1 mM NaF, 1 mM Na3VO4,
1 mM EDTA, 1 mM DTT, 0.5 mM PMSF plus the fol-
lowing protease inhibitors: 1 lg/mL pepstatin, 10 lg/mL
leupeptin and 10 lg/mL aprotinin), homogenized in a
Dounce homogenizer, and processed as described previ-
ously [18]. For Western blot analysis, the following anti-
bodies were used: rabbit anti-phospho-p53 (Abcam), rabbit
anti-Bax, mouse anti-Bcl-2, and mouse anti-GAPDH
(Santa Cruz Biotechnology, Santa Cruz, CA). In a similar
fashion, cell lysates from untreated and treated spinal
neurons were processed. Experiments were performed in
triplicate to ensure reproducibility. Values for injured,
treated and control samples (n C 3 each group) were
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compared using one-way ANOVA test. A p value of\0.05
was considered significant.
Isolation of Genomic DNA and Agarose Gel
Electrophoresis
DNAladderingisoneofthehallmarkeventsusedtoconfirm
apoptosis. Internucleosomal DNA fragmentation in the
spinalcordtissueofcontrolandspinalcordtissueadjacentto
the site of injury (caudal) in injured (1, 3, 7, 14 and 21 days
post-SCI) rats was determined using the TACS DNA Lad-
deringKit(R&DSystems,Minneapolis,MN).Briefly,DNA
extraction from tissue samples was performed as per the
manufacturer’s instructions. Tissue was minced into small
pieces and then frozen in liquid nitrogen. Frozen tissue was
powdered and lysed with the buffers provided in the kit.
DNA present in the lysed tissue was extracted using the
extraction solutions and followed by addition of sodium
acetate. DNA pellets were obtained by adding 2-propanol to
the extracted solution followed by centrifugation at
12,0009g for 10 min. DNA pellets were washed with 70%
ethanol, dried and resuspended in DNase-free water. Sam-
ples (2 lg/lane) were loaded onto 1.6% agarose gels con-
tainingethidiumbromide,electrophoresedin19TAEbuffer
and photographed on an UV transilluminator.
PCR Super Array analysis
We used the RT2 Profiler Rat Apoptosis PCR Array
(Catalog No. APRN-012, SA Biosciences, Frederick, MD).
Spinal cords of sham control and 3 weeks SCI injured rats
were removed as per the study design. Approximately 1 cm
of the segments, including the injury site, were collected
and homogenized. Total RNAs of respective tissue seg-
ments were isolated and approximately 1 lg of total RNA
from each sample was synthesized into cDNA following
manufacturer’s instructions using the iScriptTMcDNA
Synthesis Kit (Bio-Rad Laboratories, Hercules, CA). Real-
time PCR was carried out under the following conditions:
one cycle of 95?C for 10 min, and 40 cycles of 95?C for
15 s and 60?C for 1 min. Changes in gene expression were
illustrated as a fold increase/decrease. The cut-off induc-
tion for determining expression was ?2.0 or -2.0 fold
changes. Genes that fit the above criteria were considered
to be upregulated or downregulated. Nine rats were used
for each group (sham control and 3 weeks SCI) and the
RNA extracted from three individual rats was pooled and
used. We performed these experiments in duplicate.
Immunohistochemistry
Animals were sacrificed at different time points (1, 3, 7, 14
and 21 days after SCI) for studies on apoptosis under
in vivo conditions. Rats were anesthetized with ketamine/
xylazine and intracardially perfused with 4% paraformal-
dehyde in 0.1 M phosphate buffer (pH 7.4). The spinal
cords were removed and fixed for 2 h. Serial sections
(5 lm) were made from the spinal cord and stained with
H&E and Luxol fast blue for assessment of tissue mor-
phology and to determine the injury epicenter. Sections (1
section every 200 lm) obtained from 1 to 4 mm of tissue
caudal to the epicenter were used for immunohistochemical
analysis of activated apoptotic proteins. After staining with
primary antibodies (1:100 dilution), the sections were
washed three times in PBS (10 min/wash) and incubated in
goat anti-mouse or anti-rabbit HRP-conjugated secondary
antibodies (1:200 dilution). For immunofluorescence stud-
ies, the sections were washed three times in PBS (10 min/
wash) and incubated in Alexa Fluor 594 conjugated anti-
mouse secondary antibody or Alexa Fluor 488 conjugated
anti-rabbit secondary antibody (all at 1:200 dilutions) for
1 h at room temperature. Sections were then washed three
times in PBS (10 min/wash), counterstained with DAPI,
cover slipped using fluorescent mounting medium (Dako,
Carpentaria, CA) and observed under a confocal micro-
scope (Olympus Fluoview, Olympus, Melville, NY).
Negative controls (without primary antibody or using iso-
type-specific IgG) were maintained for all the samples.
Sections in which polymerase was omitted were used as the
negative control for TUNEL assay. A neuropathologist,
who was blinded to the treatment groups and study, eval-
uated the sections.
Evaluation of Cell Death by TUNEL Assay
To determine the number of apoptotic cells in the tissue
sections, paraffin-embedded tissue sections were deparaff-
inized and rehydrated. Samples were then incubated in
TUNEL reaction mixture in a humidified atmosphere for
1 h at 37?C in the dark and washed with PBS. 40, 6-di-
amidino-2-phenylindole (DAPI) staining mounting solu-
tion was used for nuclear counterstaining. Samples were
imaged with an Olympus fluorescence microscope. The
apoptotic index was defined as follows: apoptotic index
(%) = 100 9 (apoptotic cells/total cells).
Sub-Cellular Fractionation of Mitochondria
and Cytosolic Fractions
Spinal neurons were harvested, washed once with cold PBS,
and resuspended in ice-cold buffer A of Mitochondria iso-
lationkit(Sigma).CellswerethenDouncehomogenizedand
unbrokencellsandnucleiwereremovedbycentrifugationat
5009g for 5 min at 4?C. The supernatants were further
centrifugedat7,7009gfor10 minat4?Ctoobtainthe crude
mitochondrial fraction. The supernatants resulting from the
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above step were subjected to a high-speed centrifugation at
100,0009g for 1 h at 4?C to obtain the cytosolic fraction.
The crude mitochondrial pellet was resuspended in cold
buffer A and layered on the top of a 2.5 M sucrose-percoll
gradient and centrifuged at 46,0009g for 45 min at 4?C to
separate the pure mitochondrial fraction. The mitochondria
were collected and washed twice with buffer A at
7,7009g for 10 min at 4?C and lysed in the presence of cold
buffer A plus 1% Chaps for 30 min at 4?C. The lysates were
then centrifuged at 15,0009g for 10 min at 4?C to remove
insoluble debris. Protein concentration of both mitochon-
drial and cytosolic lysates was determined using the Brad-
ford dye-binding protein assay (Bio-Rad).
Comet Assay
DNA damage was assessed using the alkaline single-cell
gel electrophoresis (SCGE) ‘‘comet assay’’ method. The
comet assay has been shown to be a sensitive and reliable
measure of DNA strand breaks associated with incomplete
excision repair sites and alkali-labile sites. In the present
study, we used the OxiSelect Comet Assay Kit (Cell Bio-
labs Inc, San Diego, CA). Spinal cord tissue was processed
by re-suspension in PBS (10,000 cells/mL). 50 lL of the
cell suspension was then mixed with 500 lL of 0.5% low
melting point agarose at 37?C and 75 lL of the cell/aga-
rose mixture was transferred onto glass slides. Slides were
immersed in pre-chilled lysis buffer (2.5 M NaCl, 100 mM
EDTA, 10 mM Tris, pH 10.0, 1% Triton X-100, and 10%
Me2SO) for 1 h, followed by equilibration in Tris borate-
EDTA (TBE) buffer for 30 min. Slides were electropho-
resed in TBE at 1.5 volt/cm for 5 min and stained with
Vistra Green. Images were visualized under a fluorescence
microscope and captured with a CCD camera. Nuclei with
damaged DNA have the appearance of a comet with a
bright head and a tail, whereas nuclei with undamaged
DNA appear round with no tail. The olive tail moment is
one of the parameters that are commonly measured with
the comet assay. It represents the product of the amount of
DNA in the tail (expressed as a percentage of the total
DNA) and the distance between the centers of mass of the
head and tail regions as the measure of DNA damage. The
comet tail moment was determined and the mean ± SD of
the tail moment were obtained from 100 cells from each
treatment group.
Densitometry
ImageJ software (National Institutes of Health) was used to
quantify the mRNA and protein band intensities. Data are
represented as relative to the intensity of the indicated
loading control.
Statistical Analysis
Statistical comparisons were performed using (ANOVA)
for analysis of significance between different values using
GraphPad Prism software (San Diego, CA). Values are
expressed as mean ± SE from at least three separate
experiments, and differences were considered significant at
a P value of less than 0.05.
Results
Changes in the Expression of DNA Damage Inducing
and p53 Signaling-Mediated Apoptotic Genes After
Spinal Cord Injury
To evaluate the changes in the expression of genes
belonging to the DNA damage inducing and p53 signaling
pathways involved after injury of rat spinal cord tissues, we
performed cDNA microarray analysis using the RT2 pro-
filer PCR array. We analyzed 84 genes each belonging to
the above-mentioned two signaling pathways. Of the 84
apoptotic genes analyzed using p53 signaling RT-PCR
microarray, 6 genes showed significant changes in samples
taken 21 days after SCI (Table 1). Most of the pro-apop-
totic genes were expressed at higher levels. Many genes
related to p53 signaling, such as TP53, Cdc2 and Bag1
were upregulated after SCI (Table 1). Of the 84 DNA
damage-inducing genes, 13 genes showed significant
changes (Table 2). We observed the upregulation of DNA
damage-inducing genes ATM, ATRx, Chek1, Ogg1,
Gadd45a, TP53, Xrcc1 and PARP-I 21 days after SCI.
Along with these genes, several genes of DNA repair
enzymes were also detected. 60S ribosomal protein P1 and
RAD21 homologue genes were downregulated. These
results probably indicate that DNA damage response-
mediated p53 signaling pathway genes may be causing the
apoptosis observed during secondary pathogenesis of
spinal cord injury. To assess DNA damage response-
mediated apoptosis in injured spinal cord neuronal tissue,
Table 1 p53 signaling genes after spinal cord injury
Gene DescriptionFold up or down
regulation
Bag1Bcl2 associated gene3.01
BRCA2Breast cancer homolog22.06
Cdc2Cell division cycle2 a7.41
TP53Tumor protein p53 3.61
Cip1/Waf1 Cyclin-dependent kinase
inhibitor 1A
2.20
Ket/Tp63Tumor protein p63-4.47
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we measured DNA fragmentation using the alkaline comet
assay. This assay allows for the detection of both single-
stranded and double-stranded DNA breaks and is therefore
a highly sensitive method to directly examine the amount
of DNA damage incurred in a single cell. Our results show
that the extent of DNA damage increased from 1 day post-
SCI to 21 days post-SCI (Fig. 1a), whereas there were no
DNA strand breaks in the control spinal cord tissues. Since
the number of DNA lesions in injured spinal cord tissues
and upregulation of DNA damage-inducing molecules
were observed by comet assay and DNA damage signaling
RT-PCR array respectively, we decided to examine which
proteins were playing a major role in DNA damage after
SCI at different time points. We analyzed the tissue lysates
using western blotting for the expression of ATM, ATR,
CHK2, H2Ax, 8-OHdG, BRCA2 and Gadd45a (Fig. 1b).
Expression of ATR and ATM increased with increased
time periods. However, other molecules were highly
expressed in the injured lysates compared to control with
no significant changes over the time points (Fig. 1c). We
further decided to confirm the DNA damage caused after
SCI by carrying out immunohistochemical analysis of the
spinal cord sections of the rats from sham control and those
injured (21 days after SCI). We observed that immunore-
activity for 8-OHdG was null in sham control rats, and
positive staining for 8-OHdG red fluorescence was more
prominent in injured rats (21 days after SCI) (Fig. 1d).
Furthermore co-localization experiments on spinal cord
tissue sections adjacent to the site of injury with phos-
phorylated H2AX Ser139and NF (neuronal filament) anti-
bodies showed prominent expression of H2AX in the
nucleus of neurons of the spinal cords of the injured group.
Co-localization with neuronal factor staining confirmed the
localization of H2AX activity in the nucleus of the neurons
(Fig. 1e). Sham control spinal cord sections were negative
for phospho-H2AX Ser139fluorescence staining in the
nucleus of neurons. These results suggest that DNA dam-
age was induced in the neuronal population of injured rat
spinal cords and activation of H2AX was observed in
neurons of injured spinal cord sections.
Activated p53 Induces Apoptosis After SCI
Microarray analysis data demonstrated the upregulation of
Bax and p53 genes after spinal cord injury. To further
confirm the increase in p53 expression after spinal cord
injury, we performed immunohistochemical analysis of the
spinal cord sections of rats from sham control and injured
(21 days SCI) groups by double immunostaining. Here, we
specifically targeted the expression of p53 in three neural
cell types (oligodendrocytes, astrocytes and neurons). p53
immunoreactivity was found primarily in glial cells where
the nuclear localization of p53 is common. However, some
glial cells also contained reaction product in their cyto-
plasm. p53-positive cells were co-localized to GFAP and
Olig2, indicating that p53 is present in astrocytes and oli-
godendrocytes (Fig. 2a). In addition to these two cell types,
we identified the expression of p53 in neurons (Fig. 2a).
p53 was not detectable in sham operated control spinal
cord tissue sections. Next, we estimated protein expression
levels by western blotting. Total p53, phospho-p53 Ser392
and Bax levels were increased after 1 day and up to
21 days in injured spinal cords. On the other hand, levels of
Bcl2, which is an anti-apoptotic protein, were drastically
decreased by increased time points after injury (Fig. 2b, c).
Finally, we performed p53 transcription factor assay using
the Trans AM p53 transcription assay kit. Increased p53
transcription factor activity was observed in the nuclear
Table 2 DNA damage
inducing genes after spinal cord
injury
GeneDescription Fold up or down
regulation
ApexAPEX nuclease (multifunctional DNA repair enzyme) 1 2.08
ATMAtaxia telangiectasia mutated homolog (human) 7.93
AtrxAlpha thalassemia/mental retardation syndrome X-linked
(RAD54 homolog, S. cerevisiae)
5.74
Chek1CHK1 checkpoint homolog (S. pombe)
3.15
Cspg6/bamacan Structural maintenance of chromosomes 37.93
Gadd45a Growth arrest and DNA-damage-inducible, alpha5.35
TP53 Tumor protein p53 3.61
Xrcc1X-ray repair complementing defective repair
in Chinese hamster cells 1
4.661
Ogg18-oxoguanine DNA glycosylase 8.51
PARP1Poly (ADP-ribose) polymerase 13.61
TdgThymine-DNA glycosylase 14.79
RAD 21 RAD 21 homologue (S. pombe)-5.84
Rlpl1Ribosomal protein, large, P1-2.49
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extracts of injured spinal cord tissues of 1, 7, 14 and
21 days post-SCI (Fig. 2d). Since p53 is involved in
apoptosis, we evaluated the process of apoptosis in SCI
tissues. Figure 3a shows the co-localization of p53-positive
and TUNEL-positive cells in injured spinal cord (21 days
SCI) but there were no TUNEL-positive or p53-positive
cells detected in sham control sections. DNA laddering
assay confirmed DNA fragmentation following the injury
of spinal cords (Fig. 3b). These results confirm that p53
plays a role in induction of apoptosis after SCI.
Apoptosis is Mediated by the Mitochondrial Pathway
Accumulation of DNA damage leads to p53 activation
through phosphorylation and subsequent apoptosis in
cycling cells [19]. p53-mediated Bax activation and
induction of caspase execution has been identified in sev-
eral neurodegenerative diseases [5, 20]. p53 can mediate
mitochondrial permeabilization through direct physical
interaction with Bax and Bak. To confirm Bax-mediated
apoptosis through the mitochondrial pathway, we prepared
sub-cellular fractions from the mitochondrial and cytosolic
fractions of sham control and injured spinal cords (3 weeks
SCI). We carried out immunoblot analysis using Bax and
cytochrome c antibodies. We observed increased Bax
expression and decreased cytochrome c in the mitochon-
drial fractions of injured spinal cord tissue lysates. To
check the purity of mitochondrial fractions, we analyzed
the lysates with anti-HSP60 antibody (Fig. 3c, d). The
mechanism by which p53 influences the neuronal response
to injury is poorly understood. However, currently avail-
able data suggest that Bax mitochondrial translocation is
involved in p53-mediated neuronal cell death [21, 22]. To
confirm Bax activation in mitochondrial-mediated apop-
tosis, we performed Bax and HSP60 double immunohis-
tochemical staining. Injured spinal cord tissue sections
showed prominent expression of Bax co-localized with
HSP60 in the neuronal population of injured tissue sections
(Fig. 3e) while HSP60 and Bax co-localization was not
detected in sham controls. Furthermore, activated expres-
sion of either p53 or Bax was not detected in sham con-
trols. These results confirm the role of activated p53 in Bax
Fig. 1 DNA damage response after spinal cord injury. a Comet assay
shows accumulation of DNA-SSBs and DNA-DSBs in injured rat
spinal cords. Quantitation of damaged DNA in spinal neuronal cells
was measured by the comet assay image analysis. Columns averages
of three independent experiments with 100 cells (nuclei) analyzed per
experiment. Values are mean ± SE based on at least 10 images per
group and were replicated in 3 different rats spinal cords and comet
assays. Error bars indicate mean ± SE. Significant at P\0.05 vs.
control cells. b DNA damage response elements were detected by
Western blot analysis. Immunoblotting was done using antibodies
against ATR, ATM, CHK2, H2AX, 8-OHdG, BRCA2 and Gadd45a.
GAPDH was used as a control. Sham control, 1, 3, 7, 14 and 21 days
post-SCI spinal cord tissue lysates were used for immunoblotting.
c Quantitation of b. Error bars indicate mean ± SE. Significant at
P\0.05. d Immunohistochemical comparison of control, injured
(21 days after SCI) spinal cord sections were performed to analyze
the expression of 8-OHdG and e H2AX and NF colocalization at
the site of injury. 8-OHdG is conjugated with Alexa Fluor 594 sec-
ondary antibody. H2AX is conjugated with Alexa Fluor 594
secondary antibody and NF is conjugated with Alexa Fluor 488
secondary antibody (n = 3). Bar = 200 lm
Neurochem Res
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translocation and induction of apoptosis via the mito-
chondrial pathway. We next analyzed mitochondrial-med-
iated apoptotic pathway proteins using Western blot
analysis. Immunoblotting showed that APAF1, cleaved
caspase 9, cleaved caspase 3 and cleaved PARP-I protein
expression were increased in all injured spinal cord tissue
lysates from 3 to 21 days post-SCI. However, none of the
above-mentioned cleaved fragments were detected in sham
controls (Fig. 4a, b). Cytosolic cytochrome c induces the
activation of APAF1, and it activates caspase 9 to form
the APAF1-proteosome complex. The active proteosome
complex further acts upon pro-caspase 3 and cleaves into
an active form of caspase 3. Hence we measured the
activation of caspase 9 and 3 by colorimetric estimation.
We detected caspase 9 (Fig. 4c) and caspase 3 activity
(Fig. 4d) in all injured tissue lysates (1, 3, 7, 14 and
21 days post-SCI). These results confirm the role of
caspases 9 and 3 in p53-mediated neuronal cell death in
injured spinal cord.
Activation of p53 in Staurosporine-Treated Spinal
Neurons
Apoptosis and free radical damage are the prominent pro-
cesses involved in secondary degeneration after spinal cord
injury [23, 24]. Our in vivo experiments showed the acti-
vation of p53 in neurons after spinal cord injury. To assess
the p53 activity due to apoptosis during secondary degen-
eration, we used an apoptosis-inducing agent, staurosporine
(STP), to treat rat spinal neurons. Results of western
blotting revealed the activation of p53 and Bax in STP-
treated spinal neurons (Supplementary Figs. 1A, 1B).
p53 and phospho-p53 Ser392were significantly higher in
STP-treated spinal neurons than in untreated cells.
Fig. 2 Induction of apoptosis by activated p53 and Bax upregulation.
a Immunohistochemical analysis of p53 in astrocytes, oligodendro-
cytes and neurons of injured spinal cord tissues (21 days post-SCI).
GFAP (marker for astrocytes), Oligo2 (oligodendrocytes marker) and
MAP2 (neuronal marker) were used. GFAP, Olig2 and MAP2 are
conjugated with Alexa Fluor 594 secondary antibody and p53 is
conjugatedwithAlexaFluor
Bar = 200 lm. b Equal amounts of protein (40 lg) from sham
controls and different time points of post-SCI samples were loaded
488secondaryantibodies.
onto 10–14% gels and transferred onto nitrocellulose membranes,
which were then probed with their respective antibodies. GAPDH was
used as a positive loading control. c Quantitation of b. Error bars
indicate mean ± SE. Significant at P\0.05. (D) p53 transcription
factor assay. Sham control, 1, 7, 14 and 21 days post-SCI spinal cord
tissues were used for nuclear extracts. We checked for p53
transcription factor activity using oligonucleotides that contained
the p53 consensus DNA binding site (50-GGACATGCCCGGG-
CATGTCC-30)
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Immunocytochemical staining of p53 and phospho-p53
was observed in STP-treated spinal neurons (Supplemen-
tary Figs. 1C-D). We did not observe any expression of
phospho-p53 Ser392in control spinal neurons. Further, to
confirm the in vivo experiments representing p53 activa-
tion and Bax-mediated apoptosis in injured spinal cords,
we performed immunocytochemical analysis of spinal
neurons that were injured with STP treatment. The amounts
of red fluorescence, which is indicative of p53, and green
fluorescence, which is indicative of Bax expression, were
very slight in controls; expression increased when the cells
were treated with STP (Supplementary Fig. 2A). In the
merged images, p53 and Bax co-localization are clearly
observable in STP-treated spinal neurons. Further, to con-
firm the activation and translocation of Bax into the
mitochondria in injured spinal neurons, immunocyto-
chemical analysis was performed on spinal neurons injured
with STP. Spinal neurons were immunolabeled with anti-
HSP60 and anti-Bax antibodies. As expected, we observed
prominent yellow color, which is indicative of co-locali-
zation of HSP-60 and Bax expression, in injured spinal
neurons. In contrast, we did not observe any expression of
Bax or its localization with HSP-60 in control spinal neu-
rons (Supplementary Fig. 2B). These results are consistent
with our in vivo data and further strengthen the findings
observed in vivo.
Discussion
The most important consequence of SCI is severe nerve
injury that results from two mechanisms, primary trauma
and secondary damage. The primary trauma induces
mechanical compression, bleeding and electrolyte distur-
bances, which can lead to irreversible nerve injury. The
secondary damage is due to edema, hemorrhage, inflam-
matory reaction,lipid peroxidative reaction, energy
metabolism system disorders, ischemia and oxidative
stress, which can give rise to reversible nerve injury. We
hypothesized that after SCI, neuronal apoptosis is mediated
by p53 involving the mitochondria. To validate our
hypothesis, we analyzed the expression profiles of
Fig. 3 Post-traumatic apoptosis after spinal cord injury in vivo.
a Double labeling with terminal deoxynucleotidyl transferase dUTP
nick end labeling (TUNEL) and co-localization with p53 staining by
fluorescent-tagged secondary antibodies. TUNEL-positive and p53-
positive cells were observed in injured spinal cord sections.
Bar = 200 lm. p53 is conjugated with Alexa Fluor 594 secondary
antibody. b DNA laddering analysis of sham control, 1, 3, 7, 14 and
21 days after spinal cord injury tissue sections. * 180 bp length
DNA fragments were detected in injured tissue samples (/).
c Mitochondrial and cytosolic fractions were analyzed for Bax and
cytochrome c levels by western blotting. b-actin and HSP-60 were
used as an internal control for the cytosolic and mitochondrial
fractions, respectively. d Quantitation of c. Error bars indicate
mean ± SE. Significant at P\0.05. e Bax and HSP60 colocalization
was analyzed by immunohistochemical staining of injured spinal cord
tissues and compared with sham control sections. Bax is conjugated
with Alexa Fluor 488 secondary antibody and HSP60 and phospho-
p53 were conjugated with Alexa Fluor 594 secondary antibodies. Co-
localization of phospho-p53 and Bax was detected in injured spinal
cord tissues whereas no phospho-p53 and Bax staining was observed
in the sham controls. All experiments were performed in triplicate.
Bar = 200 lm
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apoptotic genes 21 days after SCI and found efficient
upregulation of apoptotic genes in the injured spinal cord.
Thus, the results of this study demonstrate secondary
degeneration after spinal cord injury caused by neuronal
cell death. In this study, we attempted to characterize
changes in gene expression levels in adult rats at 3 weeks
post-SCI.
Details of the underlying mechanisms of DNA damage
in the neuronal population of injured spinal cord tissues
remain unknown. The present study is important because it
strongly implicates the accumulation of DNA strand breaks
as a major triggering event for neuronal apoptosis. Here,
we also delineated the molecular mechanisms of DNA
damage-induced apoptosis upstream and downstream of
p53 in injured spinal cords. Our results show an increase in
oxidative DNA damage scores (comet assay) in the nucleus
of neuronal cells after spinal cord injury in the caudal
section. However, DNA damage in other cell groups
including glia, infiltrating leukocytes and endothelial cells
of vascular walls might also be contributing to these
changes [25]. In addition to direct DNA damage, DNA
repair mechanisms can also be impaired after SCI. In this
study, our immunohistochemistry results demonstrate
increased expression of 8-OHdG and H2AX in injured
spinal cords of the caudal region. Furthermore, RT-PCR
microarray analysis showed that expression of DNA
damage inducing genes were markedly increased after
DNA damage post-SCI. These data suggest that 8-OHdG
generation is one of the important mechanisms during
inflammatory and secondary degeneration of spinal cord
injury. The rapid accumulation of DNA strand breaks in the
neuronal population of injured spinal cord tissues places
DNA damage as an activator of the ATM/p53 regulated
apoptosiscascade. Immunoblotting
increased expression of ATM and ATR, which are DNA-
damage sensor kinases that become activated after
increasing time points of injured spinal cords and in
response to DNA damage; p53 and H2AX have been
identified as active substrates in injured tissues.
Studies in transformed cell lines have led to a proposal
that normal p53 degradation requires an intact nucleolus
and that, in most cases, pro-apoptotic activation of the p53
signaling pathway is caused by nucleolar stress [26, 27].
Also, p53 dependent apoptosis and/or p53 activation were
documented in neurons whose nucleoli were obliterated
withcamptothecin, or the
analysis showed
transcriptionalinhibitor
Fig. 4 Mitochondrial-mediated apoptotic pathway in spinal cord
sections. a Equal amounts of protein (40 lg) were loaded onto 8–14%
gels and transferred onto nitrocellulose membranes, which were then
probed with respective antibodies. Beta-actin was used as a control.
b Quantitation of a. Error bars indicate mean ± SE. Significant at
P\0.05. c, d Caspase 9 and caspase 3 activity in injured spinal
cords. Error bars indicate SEM significant at P\0.005. The
experiment was repeated twice in triplicate (n = 3)
Neurochem Res
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actinomycin D, or the knockdown/knockout of the Pol1 co-
activator transcription initiation factor-1A [28, 29]. Cell
cycle check point kinases Chk1 and Chk2 expression levels
were increased and another DNA damage recognition
marker protein, BCRA2, was elevated after spinal cord
injury. The ATM kinase can mediate neuronal cell death in
response to genotoxic stress through phosphorylation of
p53 on serine-15 and serine-20 [30]. In addition, ATM-
dependent pathways can indirectly contribute to p53
function by activating cyclin-dependent kinases and phos-
phorylation of MDM-2. In addition to the ATM/Chk2
pathway, other signalling cascades that mediate activation
of p53 after DNA damage have been identified [30]. It is
well established that p53 promotes apoptosis through
enhanced transcription of specific target genes. [31, 32]. In
p53-dependent neuronal apoptosis, the multi-domain Bcl-2
family member Bax has been identified as a major mediator
of cell death [20, 33]. p53 is known to play a key role in
determining cell death [34–37]. It is known that p53
induces death in tumor cells, which have abnormal DNA
arrangements [38], and in cells exposed to DNA damaging
agents [34]. Thus, at present, the role of p53 is considered
to be dual; one is protective of the cell while the other
induces apoptosis [39]. CNS injury is known to induce
profound glial responses, including activation and prolif-
eration. Yet, it is unclear what role might be served by p53
in the event of spinal cord injury in terms of cell protection
or death.
Bcl2, an anti-apoptotic protein [40], is known to sup-
press Bax-induced apoptosis [41, 42] by forming a
homodimer and a heterodimer [43]. In the present study,
Western blot analyses showed increased phospho-p53
Ser392and Bax expression in injured spinal cord tissues.
Immunohistochemical analysis of injured tissue sections
showed more staining for Bax and activated p53 than in the
sham control tissues. Bax immunoreactivity was mainly
co-localized with p53, suggesting that the cells were des-
tined for apoptosis. These results confirm that p53-medi-
ated Bax activation and translocation into the mitochondria
further cause cytochrome c release into the cytosol. Acti-
vation of apoptosome by cytochrome c causes activation of
caspase 3 and final PARP-I cleavage, which leads to DNA
fragmentation in neuronal cells of injured spinal cord tis-
sues. Furthermore, DNA laddering and TUNEL assays
confirmed DNA fragmentation and p53-positive and
TUNEL-positive cells in the neuronal population of the
epicentre of injured spinal cord tissues. Immunohisto-
chemical analysis of p53 in injured spinal cord tissues
revealed the presence of p53 in all neuronal cells including
neurons. In vitro experiments with STP-treated spinal
neurons also confirmed our in vivo results. Mitochondrial
translocation of Bax and p53 localization was observed
both in vitro and in vivo. Taken together, the results of the
present study identified apoptotic-signaling mechanisms at
different time points after SCI, which may lead to future
studies to establish protocols that can be used to ameliorate
secondary injury mechanisms.
Acknowledgments
assistance. We thank Shellee Abraham for manuscript preparation,
and Sushma Jasti and Diana Meister for review of the manuscript.
This study was funded by a grant from Caterpillar Inc., Peoria, IL and
OSF Saint Francis Inc., Peoria, IL (J.S.R.).
We are thankful to Noorjehan Ali for technical
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