JOURNAL OF NEUROTRAUMA 28:1– ( June 2011)

ª Mary Ann Liebert, Inc.

DOI: 10.1089/neu.2010.1561

Time-Dependent Changes in Serum Biomarker Levels

after Blast Traumatic Brain Injury

Andrea Gyorgy,1 Geoffrey Ling,4 Daniel Wingo,1 John Walker,1 Lawrence Tong,2 Steve Parks,3
Adolph Januszkiewicz,2 Richard Baumann,2 and Denes V. Agoston1

Abstract
Neuronal and glial proteins detected in the peripheral circulating blood after injury can reflect the extent of the
damage caused by blast traumatic brain injury (bTBI). The temporal pattern of their serum levels can further
predict the severity and outcome of the injury. As part of characterizing a large-animal model of bTBI, we
determined the changes in the serum levels of S100B, neuron-specific enolase (NSE), myelin basic protein (MBP),
and neurofilament heavy chain (NF-H). Blood samples were obtained prior to injury and at 6, 24, 72 h, and 2
weeks post-injury from animals with different severities of bTBI; protein levels were determined using reverse
phase protein microarray (RPPM) technology. Serum levels of S100B, MBP, and NF-H, but not NSE, showed a
time-dependent increase following injury. The detected changes in S100B and MBP levels showed no correlation
with the severity of the injury. However, serum NF-H levels increased in a unique, rapid manner, peaking at 6 h
post-injury only in animals exposed to severe blast with poor clinical and pathological outcomes. We conclude
that the sudden increase in serum NF-H levels following bTBI may be a useful indicator of injury severity. If
additional studies verify our findings, the observed early peak of serum NF-H levels can be developed into a
useful diagnostic tool for predicting the extent of damage following bTBI.

Key words: biomarkers; blast traumatic brain injury; reverse phase protein microarray

Introduction potential serum markers. It is a highly brain-specific Ca2 + -

binding protein that is most abundant in the cytosol of astroglia

A ssessing the severity of injury following traumatic

brain injury (TBI), as well as monitoring the injury
process and predicting the pathological outcome remain
major challenges in clinical practice. In addition to neuro-
monitoring and imaging, markers detectable in the peripheral
blood can offer valuable information about the severity and
progression of TBI; such markers can predict possible clinical
outcomes since blood is easily and conveniently obtainable.
The ideal serum marker of TBI should have high specificity for
the brain, high sensitivity for TBI, and rapid appearance in
serum (Bakay and Ward, 1983). Furthermore, a close corre-
lation ought to exist between the serum levels of the marker
and the extent of tissue injury.
Temporal changes in serum concentrations of S100B, neuron-
specific enolase (NSE), myelin basic protein (MBP), and neuro-
filament heavy chain (NF-H) have been studied previously in
various TBI models and clinical settings (Berger, 2006; Korfias
et al., 2009). S100B is one of the most extensively studied
(Donato, 1999). Since it has a short half-life in serum of less than
60 min (Townend et al., 2006), its levels can rise transiently after
injury, making it a good candidate as a TBI marker. NSE is a
highly neuron-specific cytosolic enzyme, and is also a fre-
quently-tested candidate biomarker (Marangos and Schmechel,
1987). MBP is a membrane-bound protein of oligodendrocytic
origin (Thomas et al., 1978), a relatively less studied marker for
TBI. However, some studies have found a good correlation be-
tween serum MBP levels and clinical outcomes (Thomas et al.,
1978; Yamazaki et al., 1995). NF-H is the heavy subunit of the
neurofilament family (Fuchs and Cleveland, 1998). This large
cytoskeletal protein is one of the most abundant proteins in
neurons, and is released from damaged as well as from dying
neurons. It has been studied as a candidate biomarker for TBI
(Anderson et al., 2008; Saljo et al., 2000).
Blast TBI (bTBI) shares clinical features of both penetrating
TBI (pTBI) and closed TBI (cTBI), while possessing unique
aspects due to the different physical forces, predominantly the

1
Department of Anatomy, Physiology and Genetics, and 4Department of Neurology, Uniformed Services University (USU), School of
Medicine, Bethesda, Maryland.
2
Division of Military Casualty Research, Walter Reed Army Institute of Research, Silver Spring, Maryland.
3
ORA, Inc., Fredericksburg, Virginia.

1
2 GYORGY ET AL.
rapid change in pressure that is transferred to the brain pa-
renchyma (Cernak et al., 2001; Mayorga, 1997; Okie, 2005).
Similar to pTBI, these forces disrupt neuronal and glial cell
bodies and their processes as well as blood vessels. Available
clinical data show a significant reduction in the volume of
various fiber tracts and an unusually early onset of edema,
probably due to the disruption of vessels and the blood–brain
barrier (Cernak et al., 2001; Kaur et al., 1997; Mayorga, 1997;
Taber et al., 2006). As in other forms of TBI, many individuals
are unconscious after exposure to blast, thus the Glasgow
Coma Scale (GCS) and other widely used assessments of
neurological severity cannot be used. Although magnetic
resonance imaging (MRI) can provide precise assessments of
the damage, it is often unavailable or contraindicated because
of potential metal fragments. Therefore, detecting and fol-
lowing changes in the levels of neuronal and glial proteins in
the peripheral blood, which correlate with the clinical out-
come, could guide early triaging and therapy.
In our efforts to characterize a large-animal model of bTBI
(Bauman et al., 2009), we determined temporal changes in
serum levels of S100B, NSE, MBP, and NF-H using reverse
phase protein microarray (RPPM) technology, which allows
the simultaneous comparison of hundreds of samples. We
found that the rapid rise in NF-H serum levels specifically
correlates with severe clinical and pathological outcomes after
bTBI, suggesting that determining temporal changes in serum
NF-H levels can help assess the severity of bTBI.
Methods
Animal model and clinical scoring
A large-animal model of bTBI that uses young adult male
Yorkshire pigs was used in our studies. The model and the
experimental conditions were described previously (Bauman
et al., 2009). The animals were exposed to different levels of
explosive blast and in turn divided into three groups ac-
cording to the severity of the injuries sustained (n = 6 in each
group, 18 animals total). A pathological scoring system was
used and coded before data analysis to avoid experimental
bias. Group 1 consisted of animals with no or unremarkable
symptoms, group 2 was moderately injured, and group 3 was
severely injured, with blood present in the trachea and/or
hemorrhage in the lungs. It was later confirmed that animals
belonging to group 1 were injured by blast overpressures of
less than 20 psi, group 2 with 20–40 psi, and group 3 with
more than 40 psi. After the proteomics analysis was com-
pleted, the code was broken to allow the identification of
those biomarkers that correlated with injury severity.
Biosamples
Blood samples were collected from the femoral vein prior to
injury and at 6, 24, 72 h, and 2 weeks after. Samples were
centrifuged at 2000g for 10 min and the supernatants were
flash-frozen and stored at - 80C until use. For printing, the
samples were thawed on ice, and diluted 10-fold in print
buffer (10% glycerol, 0.05% SDS, and 50 mM DTT in 1 · TBS).
Individual samples were then further diluted using a JANUS
Varispan Integrator and Expanded Platform Workstation
(PerkinElmer, Waltham, MA), resulting in a standard 11-point
serial dilution of each sample in triplicate in Genetix 384-well
plates (cat. no. X7022; Fisher Scientific, Pittsburgh, PA) as
described previously in detail (Gyorgy et al., 2010). The plates
were then centrifuged at 4C and 1500g for 5 min prior to
transfer into an Aushon 2470 Arrayer (Aushon Biosytems,
Billerica, MA).
Printing
Nitrocellulose-coated ONCYTE Nova glass slides (Grace
Bio-Labs, Bend, OR) were used for printing (Gyorgy et al.,
2010). The Aushon 2470 Arrayer was set-up with 16 pins and
programmed for a single deposition per dot. The spot diam-
eter was set to 250 nm with a spacing of 500 nm between dots
on the x-axis and 375 nm on the y-axis. Wash time was set at
1.2 sec without delays.
Immunochemical detection
After desiccation at 4C overnight, the slides were blocked
with 5% non-fat dry milk in 1 · TBS with 0.1% Tween-20
(TBST). Primary antibodies were diluted 1:100 in antibody
incubation buffer (0.1% BSA, Halt protease and phosphatase
inhibitor cocktail [Thermo Fisher, Waltham, MA], 1 · TBS,
and 0.5% Tween-20). The following primary antibodies were
used: S100B (cat. no. ab41548; Abcam, Cambridge, MA); NSE
(cat. no. ab53025; Abcam); MBP (cat. no. ab53294; Abcam);
and NF-H (cat. no. N4142; Sigma-Aldrich, St. Louis, MO). All
of the antibodies were tested by Western blot to validate their
specificity with swine brain samples (Supplementary Fig. 1;
see online supplementary material at http://www.liebert
online.com). The primary antibody solution was distributed
over the entire nitrocellulose surface of the slide, covered with
a cover-slip, and incubated at 4C overnight. The slides were
washed three times in 0.1% TBST for 5 min each, then incu-
bated with an Alexa Fluor 635 goat anti-mouse (cat. no. A-
31574), goat anti-rabbit (cat. no. A-31576), or rabbit anti-goat
IgG (H + L; cat. no. A-21086) secondary antibody from In-
vitrogen at a 1:6000 dilution in antibody incubation buffer for
1 h at room temperature. The slides were washed three times
in 0.1% TBST for 5 min each, and then three times again in
1 · TBS. The fluorescence signal was measured by scanning
the slides with a 633-nm-wavelength laser using a 647-nm
filter in a Scan Array Express microarray scanner (Perkin El-
mer). Data from the scan were imported into a Microsoft
Excel-based bioinformatics program for analysis.
Data analysis and bioinformatics
A Microsoft Excel-based analysis program was developed
in-house to analyze the primary data (Gyorgy et al., 2010). The
program imports intensity data from the scanner output and
calculates the total net intensity after local background sub-
traction for each spot. The background-subtracted intensity
data from the dilution series of each sample were then plotted
against dilution on a log-log graph. Regression (linear re-
gression of the log-log data) of the data was done after re-
moval of flagged data. Flagged data included spot intensities
in the saturation range or noise range, signal-to-noise ratio < 2,
or high variability between duplicate spots ( > 10–15%). The
total amount of the antigen was determined by the y-axis
intercept (i.e., extrapolating the regression to zero [the undi-
luted sample]), and marked by Y intercept (Y-cept). Such
values are log10; if a Y-cept is bigger than another by 1, it is a
difference of an order of magnitude. Graphs show the aver-
SERUM BIOMARKERS IN BLAST TRAUMATIC BRAIN INJURY 3

ages of Y-cept values obtained, with error bars indicating the
standard error of the mean at each time point.
Statistical analysis
The values for each group were compared using a two-way
mixed model for repeated measures, with severity group as a
between-subjects factor, and time as a within-subjects factor.
We followed-up with a one-way analysis of variance (ANO-
VA) at each time point. A significance level of 0.05 was used
throughout. No adjustment was made for multiple compari-
sons; thus significant findings should be considered explor-
atory and need to be confirmed by further studies.
Results
Using the RPPM technology, we determined the temporal
changes in the serum levels of four glial and neuronal markers
in a large animal model of bTBI, and ascertained whether a
correlation exists between changes in their serum levels and
the severity of the injury. We found that among the markers
tested, the early peak in serum NF-H levels showed a corre-
lation with the severity of the injury.
Serum S100B levels displayed an increase, with elevated
levels seen at 6 h after injury, and then slowly decreased with
time. More importantly, no correlation was found between
the rise in S100B levels and the animal groups (Fig. 1A). There
was also a relatively high variability of S100B serum levels
among the animals. In the immediate (6 h) sample, only in
group 1 was the S100B elevation significant compared to pre-
injury levels, while in groups 2 and 3 the elevation was just
below the level of significance. S100B levels at all later time
points were significantly higher in all groups than pre-injury
levels. Statistical analysis found no significant overall differ-
ence in S100B among groups (F2.7 = 1.57, p = 0.272), and there
was also no significant group · time interaction (F8.27 = 0.426,
p = 0.895).
In contrast to S100B, there was only a slight elevation in
NSE serum levels in all three groups of animals at 6 h post-
injury (Fig. 1B), and it was statistically insignificant. More-
over, NSE levels returned to control levels by 2 weeks after the
injury. There were no significant differences in serum levels of
NSE among any of the three groups.
Serum levels of MBP showed an immediate increase,
peaked at 6 h post-injury, and remained elevated even after 2
weeks in all animals (Fig. 1C). Of all the markers investigated,
MBP showed the greatest increase at all time points tested.
However, no correlation was found between the rise in MBP
levels and the animal groups. Again, we found no significant
overall difference in MBP levels among groups (F2.7 = 0.703,
p = 0.526), and there was also no significant group · time in-
teraction (F8.28 = 0.817, p = 0.594).
In contrast to the other markers, serum NF-H levels showed
distinct temporal patterns correlating with the severity of the
injury (Fig. 1D). A rapid increase at 6 h post-injury in serum
NF-H levels was found only in the animals in group 3. By
contrast, the animals in groups 1 and 2 displayed a steady
increase in serum NF-H levels. After careful scrutiny, it be-
came apparent that the unique, early (6 h post-injury) peak in
serum NF-H levels occurred only in animals that were se-
verely injured. NF-H levels peaked in the animals in group 2

A B

C D

FIG. 1. Time course of serum level changes of biomarkers after blast traumatic brain injury (bTBI). (A) S100B. (B) Neuron-
specific enolase (NSE). (C) Myelin basic protein (MBP). (D) Neurofilament heavy chain (NF-H). Serum samples were ob-
tained prior to and 6, 24, and 72 h and 2 weeks after injury and were analyzed by reverse phase protein microarray (RPPM).
The graphs show the averages of the values obtained, with error bars indicating the standard error of the mean at each time
point. The changes in serum levels of each marker are indicated separately for the three animal groups. Note that with
exception of NF-H, the time course of the serum marker changes investigated did not differ significantly among the three
groups.
4 GYORGY ET AL.
later, ranging from 24 h to 2 weeks after injury. In the animals
with mild injury (group 1), NF-H levels peaked at even later
time points, 72 h (1 animal) to 2 weeks (5 animals). Thus of the
four markers analyzed, only NF-H showed a temporal pattern
that closely correlated with injury severity (Fig. 2). We com-
pared the severity groups using a two-way mixed model for
repeated measures (the same as for the other markers), with
severity group as a between-subjects factor, and time as a
within-subjects factor, and found a statistically significant
group · time interaction (F8.37 = 4.81, p = 0.0001). We followed
up with a one-way ANOVA at each time point and found
significance at 6 h ( p = .047) and 2 weeks post-injury
( p = 0.047), with post-hoc tests indicating that at 6 h, NF-H was
significantly higher in group 3 (severe injury) than in group 1
(mild, p = 0.035) or group 2 (moderate, p = 0.035), and at 2
weeks, NF-H was significantly lower in group 3 (severe in-
jury) than in group 2 (moderate, p = 0.019), but not group 1
(mild, p = 0.084).
Discussion
Given the complexity of TBI pathobiology, the value of
objective biomarkers is very high. Therefore, there is an es-
pecially great need for markers that can be easily quantified in
peripheral blood for the purpose of providing information
about injury progression early after TBI (Berger, 2006; Korfias
et al., 2009).
S100B is one of the most studied markers both in animal
models and in the clinical setting (Berger et al., 2005; In-
gebrigtsen and Romner, 2003; Kleindienst et al., 2005). Several
studies have shown that serum S100B levels correlate well
with the severity of insult after brain trauma as well as is-
chemic stroke (Herrmann et al., 2000; Raabe et al., 1999; Thelin
et al., 2011, to be submitted). However, some other studies
have failed to find a direct link between increased S100B se-
rum levels and clinical outcome after mild TBI (de Boussard
et al., 2005; Stapert et al., 2005). We found that serum S100B
levels were increased significantly in all animals after 24 h.
While this increase did not show a significant correlation with
the severity of the injury, nevertheless elevated serum S100B
FIG. 2. Correlation between the time of peak levels of
neurofilament heavy chain (NF-H) and clinical outcome after
blast traumatic brain injury (bTBI). Each dot refers to an in-
dividual animal, indicating when its NF-H serum levels were
highest, and to what severity group that animal belonged to.
After completing the analysis of the serum level changes, a
pattern was revealed that the mildly-injured animals were in
group 1 (below 20 psi), moderate injuries formed group 2
(20–40 psi), and animals with severe outcomes were in group
3 (above 40 psi).
level is an indicator of CNS injury. When we perform the next
set of experiments, we will re-analyze changes in serum S100B
levels in the context of injury severity.
NSE, the cytosolic enzyme involved in neuronal glycolysis,
has also been investigated as a candidate serum biomarker for
TBI (Berger et al., 2005; Skogseid et al., 1992). Several inves-
tigators (Ergun et al., 1998; Ingebrigtsen and Romner, 2003)
found no correlation between serum NSE levels and clinical
outcome of TBI. We also found that NSE levels did not change
significantly regardless of the severity of injury. Moreover, the
slightly elevated serum NSE levels were back to pre-injury
levels at 2 weeks post-injury. The half-life of NSE in serum is
long, more than 20 h (Korfias et al., 2009). This may complicate
finding a correlation between the extent of neuronal loss and
serum NSE levels, because elevated serum levels can persist
without any additional neuronal loss.
Given that blast almost always affects the whole body,
thoracic or abdominal injuries can also contribute to the
pathophysiology of bTBI (Cernak et al., 2011). Elevated serum
levels of both S100B and NSE have been detected without TBI
(e.g., after cardiac arrest; Ekmektzoglou et al., 2007; Siman
et al., 2008). Accordingly, the origin of elevated serum levels
of NSE and S100B can be either outside of the CNS, or can
reflect secondary CNS damage, but still can be useful to pre-
dict neurological outcome (Rosen et al., 2004, 2001; Shinozaki
et al., 2009).
Studies have shown that serum MBP levels in severe head
trauma peak at 48–72 h after injury (Berger et al., 2005). We
found a similar trend in our bTBI model. All animals showed
a significant and steady increase in serum MBP levels, al-
though the maximum serum MBP values were seen at 24 h
after injury. However, we did not find a correlation between
serum MBP levels and clinical outcome at any time point
tested. As was the case for S100B serum levels, a lack of sta-
tistical significance in the difference between the groups does
not rule out the possibility of a correlation between MBP
levels and injury severity; however, our data do not provide
sufficient evidence to prove this point. However, we will re-
evaluate potential correlations between injury severity and
MBP levels in future experiments. The elevated serum MBP
levels we found are consistent with the available clinical
findings using in vivo imaging. Patients suffering from bTBI
have shown significant reductions in some white-matter
tracts, indicating damaged myelin (Levin et al., 2010).
Axonal injury results in the release of various NF proteins
(Anderson et al., 2008). Among them, phosphorylated NF-H
(pNF-H) has shown a correlation with the severity of injury
after various forms of TBI (Petzold, 2005). Our finding that
shows a rapid increase in serum NF-H levels in animals with
severe bTBI suggests extensive and rapid axonal loss. This is
consistent with the increased levels of MBP and the in vivo
imaging data mentioned above. Whether pNF-H also dis-
plays a significant and rapid increase remains to be tested in
future experiments. Overall, the increases seen in serum NF-H
and MBP levels suggest that axonal injury may be an im-
portant early component of the pathobiology of bTBI.
In conclusion, we found that the early peak in serum NF-H
levels may indicate severe injury and poor outcome after
bTBI. Determining the temporal pattern of changes in serum
NF-H levels along with S100B and MBP levels can provide
much needed information for triaging and monitoring out-
come following bTBI. Furthermore, our studies show the
SERUM BIOMARKERS IN BLAST TRAUMATIC BRAIN INJURY
importance of serial sampling and assaying serum levels of
these glial and neuron-specific proteins, as the temporal pat-
tern of changes can be an important indicator of the outcome
of bTBI. We set out to find any possible association between
biomarker alterations and the severity of blast injury. Statis-
tical analysis showed that NF-H levels in the serum at 6 h and
2 weeks were different in the severely-injured and the mod-
erate or mild injury groups. However, due to the exploratory
nature of our work, further experiments need to be conducted
to address the complex relationship between the temporal
changes seen in serum biomarkers and severity and outcome
after bTBI.
Acknowledgments
The work was funded by DARPA PREVENT. We thank
Mrs. Carla Olsen for her help with the statistical analysis, and
Ms. Nicole Draghic and Ms. Alaa Kamnaksh for editorial
support.
Author Disclosure Statement
No conflicting financial interests exist. The views, opinions,
and/or findings contained in this article are those of the au-
thors and should not be interpreted as representing the official
views or policies, either expressed or implied, of the Defense
Advanced Research Projects Agency or the Department of
Defense.
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Address correspondence to:
Denes V. Agoston, M.D., Ph.D.
Department of Anatomy, Physiology and Genetics
Program in Neuroscience
Neurosurgery Program National Capital Consortium
School of Medicine, Uniformed Services University (USU)
4301 Jones Bridge Road
Bethesda, MD 20814
E-mail: vagoston@usuhs.mil