CSF-Biomarkers in Olympic Boxing: Diagnosis and Effects
of Repetitive Head Trauma
Sanna Neselius1,2*, Helena Brisby1,2, Annette Theodorsson3,4, Kaj Blennow5,6, Henrik Zetterberg5,6,
1Department. of Orthopaedics, Sahlgrenska University Hospital, Gothenburg, Sweden, 2Institution For Clinical Sciences, The Sahlgrenska Academy at University of
Gothenburg, Sweden, 3Neurosurgery Section, University Hospital in Linko ¨ping, Linko ¨ping, Sweden, 4Institution of Clinical and Experimental Medicine, Linko ¨ping
University, Linko ¨ping, Sweden, 5Clinical Neurochemistry Laboratory, Department of Psychiatry and Neurochemistry, Sahlgrenska University Hospital, Gothenburg,
Sweden, 6Institute of Neuroscience and Physiology, The Sahlgrenska Academy at University of Gothenburg, Gothenburg, Sweden, 7Geriatric Section, University Hospital
in Linko ¨ping, Linko ¨ping, Sweden
Background: Sports-related head trauma is common but still there is no established laboratory test used in the diagnostics
of minimal or mild traumatic brain injuries. Further the effects of recurrent head trauma on brain injury markers are
unknown. The purpose of this study was to investigate the relationship between Olympic (amateur) boxing and
cerebrospinal fluid (CSF) brain injury biomarkers.
Methods: The study was designed as a prospective cohort study. Thirty Olympic boxers with a minimum of 45 bouts and 25
non-boxing matched controls were included in the study. CSF samples were collected by lumbar puncture 1–6 days after a
bout and after a rest period for at least 14 days. The controls were tested once. Biomarkers for acute and chronic brain injury
Results: NFL (mean 6 SD, 5326553 vs 135651 ng/L p=0.001), GFAP (4966238 vs 2476147 ng/L p,0.001), T-tau (58626
vs 49621 ng/L p,0.025) and S-100B (0.7660.29 vs 0.6060.23 ng/L p=0.03) concentrations were significantly increased
after boxing compared to controls. NFL (4026434 ng/L p=0.004) and GFAP (3696113 ng/L p=0.001) concentrations
remained elevated after the rest period.
Conclusion: Increased CSF levels of T-tau, NFL, GFAP, and S-100B in .80% of the boxers demonstrate that both the acute
and the cumulative effect of head trauma in Olympic boxing may induce CSF biomarker changes that suggest minor central
nervous injuries. The lack of normalization of NFL and GFAP after the rest period in a subgroup of boxers may indicate
ongoing degeneration. The recurrent head trauma in boxing may be associated with increased risk of chronic traumatic
Citation: Neselius S, Brisby H, Theodorsson A, Blennow K, Zetterberg H, et al. (2012) CSF-Biomarkers in Olympic Boxing: Diagnosis and Effects of Repetitive Head
Trauma. PLoS ONE 7(4): e33606. doi:10.1371/journal.pone.0033606
Editor: Yu-Feng Zang, Hangzhou Normal University, China
Received December 7, 2011; Accepted February 13, 2012; Published April 4, 2012
Copyright: ? 2012 Neselius et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits
unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: This study has been funded by the Marianne and Marcus Wallenberg Foundation, Sweden; ALF Grants, County Council of Va ¨stra Go ¨taland, Sweden;
Sahlgrenska University Hospital, Gothenburg, Sweden; Gothenburg Medical Society, Sweden; ALF Grants, County Council of O¨stergo ¨tland, Sweden and The
Swedish Research Council. The funders had no role in study design, data collection and analysis, decision to publish or preparation of the manuscript.
Competing Interests: The authors have declared that no competing interests exist.
* E-mail: firstname.lastname@example.org
Violence against the head is a common event in a number of
different sports and may result in sports-related Traumatic Brain
Injury (TBI). In about 26% of all patients with head injuries
seeking medical help at the emergency room the trauma was
reported to be sports-related . Terms used for mild, acute head
injuries are concussion or mild traumatic brain injury (MTBI).
The diagnosis of MTBI today is established by symptoms and
clinical evaluation including a detailed neurological examination
with balance testing and cognitive function [2,3]. The manage-
ment of mild head injuries is admission for observation and/or
discharging after a normal computer tomography (CT) scan .
No information about the size or grade of the MTBI can be given
and the time point when to return to sport is commonly decided
using the Zurich 2008 Consensus Statement Return-to-Play
protocol . One problem with this protocol is that it is only
useful when the patients have symptoms with a certain severity
grade (e.g. clearly classified as a concussion).
Relatively little is further known about the late effects of
multiple MTBIs. Epidemiological and animal studies have
suggested association between repeated MTBIs/concussions and
long-term consequences in form of chronic traumatic brain injury
(CTBI) [6,7,8]. Several concussions have been demonstrated to
lead to slower recovery  and young brains have been described
to be more sensitive and needing a longer time for recovery after a
MTBI compared to adults .
In association to the lack of objective diagnostic tools for MTBI
the acute and long-term effects of olympic (amateur) boxing on the
brain are debated. Several studies have failed to prove any signs of
PLoS ONE | www.plosone.org1 April 2012 | Volume 7 | Issue 4 | e33606
brain damage associated with olympic boxing [11,12,13] whereas
the harmful long-term effects of professional boxing in form of
dementia pugilistica have been known since 1928 [14,15]. In
boxing, acute TBI can be caused by structural brain injuries such
as subdural haematoma, intracerebral haemorrhage or possibly by
repeated MTBI/concussions . MTBI may be caused by
knock-out (KO) with loss of consciousness or by the cumulative
effect of translational and rotational punches to the head .
These types of forces can result in cortical damage and diffuse
axonal injury (DAI), which eventually may lead to CTBI [18,19].
The relation between CTBI and Alzheimer’s disease is debated.
Neuropathologically, both conditions are characterized by neuro-
fibrillary tangles and amyloid-containing plaques but the location
and relative abundance of the changes differ; CTBI patients
generally have more tangles than plaques preferentially involving
the superficial cortical layers .
Presently, no objective diagnostic test, such as radiological
or laboratory measurements to diagnose, grade and monitor
TBI or early stages of CTBI, is in clinical use. CT (computed
tomography), MRI (magnetic resonance imaging) and EEG
(electroencephalography) are not sensitive enough and neuropsy-
chological tests without a baseline measurement are difficult to
interpret [11,12,13]. Analysis of CSF biomarkers can hopefully
help us understand the pathology of a MTBI at the cellular level
and may also have a role in clinical practice. To identify a reliable
tool for grading of MTBI by individuals and further to use this tool
in the evaluation of recovery, would be useful in return to sport
Biomarkers for brain damage include neurofilament light
protein (NFL), a marker of subcortical myelinated axons ,
total tau (T-tau), a marker of cortical axons [22,23], tau phos-
phorylated at threonine 181 (P-tau181), a marker of tangle
pathology , heart-type fatty acid binding protein (H-FABP),
a marker of grey matter neurons , glial fibrillary acidic protein
(GFAP)  and S-100B as markers of astroglial cells [27,28] and
the 42 amino acid isoform of amyloid b (Ab1–42), marker of
plaque pathology .
In the present study the relation between CSF biomarkers and
boxing exposure in elite olympic boxers were investigated, both in
the acute phase (within 6 days after a bout) and after a resting
period (minimum 14 days), with the aim to search for tests to
diagnose and monitor the effects of repetitive head trauma as in
The study was designed as a prospective prognostic follow-up
study. Thirty olympic boxers competing at high national and/or
international level were compared to 25 healthy, age-matched
controls. All boxers had completed at least 45 bouts. This number
was based on the regulation of the National Boxing Federation
demanding an examination with MRI, CT or EEG every 50 bouts.
The controls consisted of friends or relatives to the boxers, aimingto
get controls with similar social background and education level.
Exclusion criteria were athletes at elite level in sports where head
trauma may occur, e.g., soccer, ice hockey and martial arts.
The regional ethical review board at Linkoping Health
University, Sweden approved the study. Written informed consent
was obtained from all participants.
Questionnaire design and neurological examination
All participants filled in a questionnaire about medical history,
medication, education, present occupation, information about
previous concussions and quantification of alcohol and drug
intake. Previous sports career was reported, to identify those who
had trained in sports with risk of TBI. The questionnaire included
a 10-question survey regarding previous and current symptoms of
head and neck injuries based on a previous study . The
number of symptoms that had worsened over the last 5–10 years
was added in a score. The boxers reported about their boxing
career; fighting record, number of knock-out (KO) losses, number
of Referee Stopping Contest losses due to several hard punches to
Head (RSC-H), present weight class, duration of career, age at
career start and age at first bout [15,29]. Boxers gave an account
for total amount of bouts the last week prior testing (1–3 bouts) and
estimated these bouts as easy (1), intermediate (2) or tough (3).
Three boxing experts independently (without any knowledge of
the CSF biomarker concentrations) graded the boxers considering
head trauma during total boxing career, 1 to 5 (1 is a boxer that
has a low risk to receive blows to the head, according to boxing
style, skills and the skills of the opponents. 5 is a boxer with high
risk to receive repeated blows to the head). The total amount of
bouts the last week before test A, the boxers own grading of the
bouts and the mean of the expert grading were added in a score.
This score was named ‘‘Boxing Exposure’’. The aim was to
calculate the total MTBI risk prior testing.
All participants underwent a neurological examination prior
to lumbar puncture . The neurological examination protocol
included anamnestic questions about concussion symptoms, a
general somatic status (general condition, examination of mouth
and throat, heart, blood pressure, abdominal palpation, peripheral
circulation and skin status) and a neurological status (orientation,
alertness, speech function, cranial nerves 1–12, motor skills,
balance, coordination, gate, sensibility testing and testing of
reflexes). Magnetic resonance imaging (MRI) of the brain and
neuropsychological testing (including among others short and long
time memory, mental speed, recollection and cognitive testing)
were performed in all participants without any structural injuries
(haemorrhages, subdural haematomas) or other major findings
observed. Detailed results of these investigations will be presented
in a separate paper.
CSF sample collection
The LP was performed at daytime, between 10 a.m. and 3 p.m,
with the study objects in sitting position or laying on one side. For
the first 18 objects a Quincke Type Point spinal needle (22 Gauge)
were used, but since a few of the study objects suffered from
postspinal headache, the needle was changed to a Sprotte (24
Gauge). Thereafter no more postspinal headache occurred. For
each study object 5–10 ml CSF was collected in a polypropylene
tube (Sarstedt, Nu ¨mbrecht, Germany), gently mixed to avoid
gradient effects, aliquoted and stored at 280uC pending analysis.
LPs were performed twice in the boxers: First LP 1–6 days after a
bout (test A) and the second without exposure to bouts or training
with blows to head for at least 14 days (test B). The control subjects
underwent one LP.
NFL and GFAP were analysed using previously described
ELISA methods [21,30]. The detection limit of the NFL ELISA
was 125 ng/L. CSF total tau (T-tau), tau phosphorylated at
threonine 181 (P-tau181), and Ab1–42 levels were determined
using xMAP technology and the INNOBIA AlzBio3 kit (In-
nogenetics, Zwijndrecht, Belgium) as previously described . S-
100B was determined by an electrochemoluminescence immuno-
assay using the Modular system and the S100 reagent kit (Roche
Diagnostics). H-FABP was measured using a commercially
CSF-Biomarkers in Olympic Boxing
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available ELISA method (Hycult Biotechnology, Uden, The
Netherlands), following the instructions from the manufacturer.
Intra-assay coefficients of variation were ,10% for all assays.
All samples were analysed on one occasion to eliminate any inter-
Differences between boxers and controls for the marker
variables were tested by Student’s t-test except for NFL where
the non-parametric Mann Whitney U test was used (since many
of the samples were below the detection limit ,125 ng/L, a
parametric test could not be used.) For the boxers, differences
between time point A and B were compared using a paired sample
T-test for all biomarkers except NFL where the non-parametric
Wilcoxon signed rank test was used. For the boxers, differences
between time point A and B were compared using a paired test.
Regression analysis was used as an exploratory tool to explain
variation of the marker values as a function of different factors.
Bayesian Model Selection was used to identify the best predictive
model . Strength of association between the markers and
‘‘Boxing Exposure’’ was assessed by Spearman’s rank correlation.
Statistical analysis was carried out with SPSS 17.0 and R 2.10.
Two participants with complications after the lumbar puncture
(back pain and headache, respectively) and two without compli-
cations declined follow-up.
Questionnaire design and neurological examination
The questionnaire about medical and social history and the 10-
question survey were similar between boxers and controls (table 1).
It is to be observed that none of the boxers suffered from loss of
consciousness during their last bout before test A. Only one of the
boxers reported concussion related symptoms after the bout (in
this case headache) at the clinical examination, but the medical
and neurological examination was normal in all subjects, GCS 15.
There was no correlation between age or the risk factors listed in
table 2 and brain injury markers when using a multiple regression
model but ‘‘Boxing Exposure (BE)’’ gave a positive NFL (test A)
correlation R=0.396, p=0.030 (fig. 1).
Biomarkers for neuronal injury
The boxers had elevated concentrations of NFL at test A and B
compared to controls (Table 3, fig. 2). Only five of 30 boxers had
NFL below the detection limit of 125 ng/L after a bout, which is
considered normal for this age group  and the rest had
.125 ng/L. One of the controls had a concentration of 380 ng/
L, all the others below 125 ng/L. At follow-up, 13 of 25 boxers
had NFL.125 ng/L (fig. 2). Regression analysis for test A showed
that NFL level increased by 147 ng/L per day between days 1–6
after a bout (61 SE 67.0), t=2.190, p=0.037 (fig. 3). The two
boxers who had the highest values at the first test, 2340 ng/L and
2480 ng/L, were tested 5 days after a bout. After a resting period
of 14 days, the concentration was 125 and 1600 ng/L, respectively
(fig. 2). Their Boxing Exposures (BE) were 1 and 2 tough bouts and
expert score 4.0 and 5.0, respectively. The only boxer reporting
sequelae (headache) had ‘‘BE’’: 3 bouts, tough and mean expert
score of 3.7. Test A performed a day after the last bout revealed a
NFL concentration of 600 ng/L, which increased to 1780 ng/L
15 days later (fig. 2). In total, 7 of the boxers with pathological
concentrations of NFL at test A had even higher values at follow-
up (fig. 2). Interestingly one of these had not been boxing for 360
Concentrations of T-tau were significantly higher at test A in
boxers compared to controls (Table 3, fig. 2). The concentrations
had normalized at follow-up, although one of the boxers with high
concentrations of NFL (2480 ng/L) and GFAP (960 ng/L) after a
bout also had high concentration of T-tau at the follow-up,
121 ng/L (fig. 2). Regression analysis showed that the concentra-
tion of T-tau increased by 8.8 ng/L per day (61 SE 1.7),
t=22.477, p=0.019 in the time span 1 to 6 days after a bout
With regards to H-FABP, no significant differences were found
between boxers and controls (Table 3).
Biomarkers of astroglial injury
The boxers had elevated concentrations of GFAP at both test A
and B compared to controls (Table 3, fig. 2). All controls had
GFAP levels between 90–380 ng/L except the subject with
Table 1. Baseline details of boxers and controls.
Age, years 22 (17–34)22(17–30)
Primary School13% 20%
High School67% 64%
Other sports trained where trauma
against head can occur (years).
10- question survey2
Mean number 6 SD1.6061.831.661.71
,once per week50%56%
Once per week3%20%
.once per week7%8%
Drugs (marijuana, hashish)0%12%
1One of the boxers was born without the smell sense and had been evaluated
2Neuropsychological intervention with 10 different symptoms of head and neck
injury based on a previous study . Worsening of the number of symptoms
the last 5–10 years was evaluated.
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elevated concentration of NFL (fig. 2). This individual had a
GFAP concentration of 820 ng/L, which is considered abnormal
for this age group . Eighteen of 30 boxers had GFAP
concentrations $410 ng/L (the value calculated by mean con-
centration of the controls, without the outlier, plus 2SD) at test A
including three boxers that were lost to follow-up. Seven boxers
still had GFAP concentrations $410 ng/L at follow up (fig. 2).
One boxer had a GFAP concentration of 70 ng/L at the first test
and 560 ng/L at follow up (fig. 2). In between the two tests, the
boxer had fought 84 bouts. Test A by the boxer with the highest
value, 1020 ng/L, was taken 2 days after a series of 3 bouts, scored
easy and with a mean expert score of 2.0. The corresponding NFL
concentration was 930 ng/L. The two boxers with the highest
NFL concentrations at test A, had corresponding GFAP of 560
and 960 ng/L (test A) and 290 and 520 ng/L (test B), respectively.
Concentrations of S-100B were significantly increased after a
bout, but normalized at follow up, compared to controls (Table 3,
Markers of neurofibrillary tangle and plaque pathology
No significant differences between boxers and controls were
found for P-tau and Ab1–42, although the variability for Ab1–42
was larger in the boxer group, 238% to +100%, mean +16%
6SD 37 (fig. 4). The boxer with the lowest Ab1–42 concentration
at test A and B showed no pathological CSF concentrations of
NFL or GFAP. This boxer had a low education and career level,
had trained soccer for 8 years and started boxing 8 years ago at an
age of 12 years. The boxing career included 40 diploma bouts and
51 normal bouts. Five of the boxers showed 16–43% decreases of
Ab1–42 at the follow up whereas 2 of the boxers had increased
concentrations (15 and 76% respectively).
Figure 1. Cerebrospinal fluid concentrations of NFL in boxers
test A correlate with Boxing Exposure. Boxing Exposure is a score
that was constructed to calculate the total risk for traumatic brain injury
before testing. It consists of three factors: 1. The total amount of bouts
the last week before test A (1–3), 2. The boxers own grading of the
bouts (easy (1), intermediate (2) or tough (3) and 3. The mean of the
expert grading (3 boxing experts graded the boxers considering head
trauma during total boxing career, 1 to 5). The results of these three
factors were added in the boxing exposure score. Neurofilament light
protein (NFL) analysed in cerebrospinal fluid (CSF) after bout (test A)
correlated with Boxing Exposure, R=0.396, p=0.030.
Table 2. Boxer’s details and risk factors for brain injury.
30 boxers, mean 22, range 17–34
26 boxers, mean 24, range 17–34
AGE, WHEN THE
Mean 13.9 (median 14) Range 7–19
AGE AT FIRST BOUT
Mean 14.6 (15) 10–19
Mean 7.2 (8) 3–13
Mean 17.5 (10) 0–57
Test A Mean 74 (61) 47–168
Test B Mean 92 (79) 47-.200
Test AMean 70 (71) 25–92
Test B Mean 68 (70) 25–92
One 8 (27%)
One 5 (17%)
WEIGHT CLASS (kg) Mean 70.2 (69) 54–91
Defensive boxer 7%
Mean score #2.0 7%
Mean score 2.1–3.974%
Mean score $4.020%
DAYS SINCE LAST
Test AMean 2.7 (2) 1–6
Test B Mean 148 (26) 14–760
Scoring of last bout6
20%, Easy 47%, Intermediate33%, Tough**
Number of bouts7
40%, 1 bout40%, 2 bouts20%, 3 bouts**
3% (1 boxer)**
11–6 days after a bout;
2A rest period of a minimum of 14 days;
3Boxing at age 10–14 years without hard punches;
4Referee Stops Contest due to hard blows against head;
5Three experts graded the boxers 1 to 5, independently, (from low to high head
trauma exposure considering total boxing career);
6The boxers scored their last fight as easy, intermediate or tough;
7Number of bouts in a row (maximum one per day) for the test A;
8If a boxer experienced some sequelae after the last bout;
**Boxers with increased risk for MTBI.
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This study has gathered the largest sample so far of amateur elite
boxers and matched controls to examine a possible relationship
between MTBI in amateur boxing and CSF biomarkers.
Although only one of the boxers had self-reported symptoms of
a possible concussion and the clinical examination was normal in
all the boxers, the data demonstrate that concentrations of NFL,
GFAP, S-100B and T-tau in CSF were increased within 6 days
after a bout in more than 80% of the amateur boxers indicating
acute axonal and neuronal damage. NFL, GFAP and T-tau are
specific markers for damage of the central nervous system and
increased concentrations of NFL [21,33] and GFAP  have
previously been found both after acute and chronic brain injuries
caused by different types of trauma. Both NFL and GFAP further
remained significantly elevated after a resting period of at least 14
Table 3. Biomarker concentrations in CSF.
Boxer Test A1N=30
Boxer Test B2N=26
A vs. CA vs. B B vs. C
GFAP 496(70–1020)238367(170–600)113 244(90–820)145
FABP 407(108–1089)208 334(40–769)195 458(67–1383)2710.45 0.070.07
0.76(0.34–1.68)0.290.63(0.33–0.99)0.160.60(0.30–1.16)0.230.03 0.016 0.67
Ab1–42 306(191–411)52294(178–423)54297(231–362)390.43 0.370.83
T-Tau 58(25–132)2549(19–121)2145(24–95)170.0250.024 0.39
P-Tau 21(9–38)7 22(9–43)823(14–40)60.210.09 0.68
1Test A: 1–6 days after last bout;
2Test B: No boxing for at least 14 days. Statistical analyses were performed with parametric methods for all biomarkers except NFL where non-parametric method was
used due to values below the detection limit.
Figure 2. The individual change of CSF biomarker concentrations for boxers vs controls. Cerebrospinal fluid (CSF) was collected from
controls once. In boxers the CSF was collected 1–6 days after a bout (A) and after a rest period of at least 14 days (B). The figure illustrates the
individual change of neurofilament light protein (NFL), glial fibrillary acidic protein (GFAP), S-100B and total-tau (T-tau). NFL in all controls expect one
(380 ng/L) were below the detection limit of 125 ng/L, expressed as 125 ng/L on the chart. All controls had GFAP levels between 90–380 ng/L except
the subject with elevated concentration of NFL.
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days (test B) in the boxers compared to the controls in the present
study. Most of the boxers with increased NFL and GFAP
concentrations at test B had fought many bouts, both before test
A, and between the two tests, which may have resulted in
a cumulative effect. One previous study has analysed CSF
biomarkers in relation to boxing, where 14 boxers compared to
10 controls showed elevated concentrations of NFL, GFAP and T-
tau 7–10 days after a bout. Only NFL remained elevated at a 3
month follow-up and correlation was found between CSF
concentration of NFL and an injury severity score . Also in
the present study, a correlation was found between a composed
boxing exposure index and NFL, but construction of such an
index is difficult since the development of a possible brain injury
could potentially be related to all the risk factors listed in table 2.
Concentrations of NFL and T-tau gradually increased with time
during the first 6 days after trauma. These results are in
accordance with previous findings in patients with TBI . No
concentration peak for GFAP was found, but the boxer with the
reported concussion had among the highest concentration levels of
GFAP one day after the bout and the levels had decreased from
960 to 500 ng/L at follow up, 15 days after the bout. More studies
are needed to investigate if NFL and GFAP correlate with the size
Our study also revealed higher concentrations of S-100B after a
bout compared to controls.
S-100B is a calcium binding protein physiologically produced
and released by astrocytes and other glial cells in the central
nervous system (CNS) . Outside the nervous system it can be
found in adipose tissue, muscle and skin . S-100B increases
after brain injury and remains elevated for up to 5 days in CSF,
with a peak at day one . The concentrations have been
observed to correlate with brain injury severity . In serum, S-
100B increases after MTBI  and S-100B levels have also been
found to rise after physical activity such as marathon running. S-
Figure 3. CSF biomarker concentrations vs days in test A. Cerebrospinal fluid concentrations of NFL, GFAP, T-tau and S-100B from the boxer
group after bout (test A) plotted vs time in days after their last bout. For NFL and T-tau increasing concentrations are seen with time. The opposite is
observed for S-100B and no relationship was seen between GFAP and time when the samples were collected.
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100B released from skeletal muscle has a relatively short half-life in
serum and the levels are back to normal levels within 20 hours
. To our knowledge no studies have shown transport of S-
100B from serum to CSF, why analysis of S-100B in CSF most
likely reflects the true cerebral S-100B concentration . The
role of released S-100B after TBI is not clearly understood but it
might have both neurotrophic and neuroprotective functions, or
simply reflect injury-related release .
Little is known about the dynamics of Ab1–42 in CSF but
recently the concentrations of Ab1–42 were demonstrated to
correlate with neurological status after acute brain injury. The
concentrations were consistent between different sampling occa-
sions in healthy patients but decreased after a brain injury and
increased when the neurological status improved . Therefore,
even though no statistic differences were found, one explanation
for the relatively large variation in Ab1–42 concentrations
between test A and B in some of the boxers may be a traumatic
brain injury at some stage. In a similar manner the large variation
of P-tau with indications of gradual increase in the boxers may be
an early sign of neurofibrillary tangle build-up.
The strength of this study is the very well matched baseline
parameters for boxers versus controls. The only difference was a
longer career of other sports where trauma against head occurs in
controls compared to boxers (24% vs 0%) (table 1). Interestingly,
the only control subject with elevated levels of NFL and GFAP was
among those who had a previous sport career including head
trauma. A previous study on CSF biomarkers in soccer players did
not find any evidence of TBI in that group . A limitation of
this study is the variation of time points for CSF sampling. The
ideal had been to have test A and B collected at the same time
points for all participants. It would also have been preferable with
a longer rest period before the B sampling. This was not possible
since we had to adapt the samplings to the boxers’ schedules in
order to perform the study.
In conclusion, this study shows that the repetitive head trauma
occurring in olympic boxing may induce changes in CSF NFL,
GFAP, T-tau and S-100B, even without anamnestic or clinical
symptoms of a concussion or traumatic brain injury. These
changes suggest minor central nervous system injuries. It seems
that most of the acute injuries can recover with rest but without an
appropriate rest period there might be a risk for cumulative injury.
The length of the rest period needed seems either to be individual
or is correlated to the size of the injury.
Further studies are needed to evaluate if nervous system injury
biomarkers in CSF may be useful as an evaluation tool in clinical
praxis in the diagnosis and grading of a concussion/MTBI and as
part of return to sport guidelines. Future studies of interest include
closer monitoring of boxers at different early time-points after
repeated head trauma at bouts, long-time follow-up of boxers
and also monitoring of CSF biomarkers in patients attending
emergency departments due to a concussion where the clinical
examination is normal.
The authors want to thank Lisbeth Hja ¨lle for her helpful assistance with
this study, including laboratory work and guiding of study subjects.
Conceived and designed the experiments: SN HB AT KB HZ JM.
Performed the experiments: SN AT KB HZ JM. Analyzed the data: SN
HB KB HZ JM. Contributed reagents/materials/analysis tools: SN HZ
KB JM. Wrote the paper: SN HB AT KB HZ JM.
1. Browne GJ, Lam LT (2006) Concussive head injury in children and adolescents
related to sports and other leisure physical activities. Br J Sports Med 40:
Jagoda AS (2010) Mild traumatic brain injury: key decisions in acute
management. Psychiatr Clin North Am 33: 797–806.
Putukian M (2011) The acute symptoms of sport-related concussion: diagnosis
and on-field management. Clin Sports Med 30: 49–61, viii.
Ingebrigtsen T, Romner B, Kock-Jensen C (2000) Scandinavian guidelines for
initial management of minimal, mild, and moderate head injuries. The
Scandinavian Neurotrauma Committee. J Trauma 48: 760–766.
McCrory P, Meeuwisse W, Johnston K, Dvorak J, Aubry M, et al. (2009)
Consensus statement on Concussion in Sport–the 3rd International Conference
on Concussion in Sport held in Zurich, November 2008. J Sci Med Sport 12:
Omalu BI, Bailes J, Hammers JL, Fitzsimmons RP (2009) Chronic traumatic
encephalopathy, suicides and parasuicides in professional American athletes: the
role of the forensic pathologist. Am J Forensic Med Pathol 31: 130–132.
Omalu BI, Hamilton RL, Kamboh MI, DeKosky ST, Bailes J (2010) Chronic
traumatic encephalopathy (CTE) in a National Football League Player: Case
report and emerging medicolegal practice questions. J Forensic Nurs 6: 40–46.
Kane MJ, Angoa-Perez M, Briggs DI, Viano DC, Kreipke CW, et al. (2011) A
mouse model of human repetitive mild traumatic brain injury. J Neurosci
Methods 203(1): 41–49.
9. Guskiewicz KM, McCrea M, Marshall SW, Cantu RC, Randolph C, et al.
(2003) Cumulative effects associated with recurrent concussion in collegiate
football players: the NCAA Concussion Study. JAMA 290: 2549–2555.
10. Field M, Collins MW, Lovell MR, Maroon J (2003) Does age play a role in
recovery from sports-related concussion? A comparison of high school and
collegiate athletes. J Pediatr 142: 546–553.
11. Haglund Y, Bergstrand G (1990) Does Swedish amateur boxing lead to chronic
brain damage? 2. A retrospective study with CT and MRI. Acta Neurol Scand
12. Haglund Y, Edman G, Murelius O, Oreland L, Sachs C (1990) Does Swedish
amateur boxing lead to chronic brain damage? 1. A retrospective medical,
neurological and personality trait study. Acta Neurol Scand 82: 245–252.
13. Haglund Y, Persson HE (1990) Does Swedish amateur boxing lead to chronic
brain damage? 3. A retrospective clinical neurophysiological study. Acta Neurol
Scand 82: 353–360.
14. Martland H (1928) Punch Drunk. JAMA 91: 1103–1107.
15. Kaste M, Kuurne T, Vilkki J, Katevuo K, Sainio K, et al. (1982) Is chronic brain
damage in boxing a hazard of the past? Lancet 2: 1186–1188.
16. Charnas L, Pyeritz RE (1986) Neurologic injuries in boxers. Hosp Pract (Off Ed)
21: 30–31, 34–39.
17. Viano DC, Casson IR, Pellman EJ, Bir CA, Zhang L, et al. (2005) Concussion in
professional football: comparison with boxing head impacts–part 10. Neurosur-
gery 57: 1154–1172; discussion 1154–1172.
Figure 4. Ab1–42 shows a larger variation in the boxers vs
controls. Cerebrospinal fluid (CSF) was collected from the controls
once. The boxers were tested 1–6 days after a bout (A) and after a rest
period without exposure to bouts or training with blows to head for at
least 14 days (test B).
CSF-Biomarkers in Olympic Boxing
PLoS ONE | www.plosone.org7 April 2012 | Volume 7 | Issue 4 | e33606
18. Adams JH, Doyle D, Ford I, Gennarelli TA, Graham DI, et al. (1989) Diffuse Download full-text
axonal injury in head injury: definition, diagnosis and grading. Histopathology
19. Topal NB, Hakyemez B, Erdogan C, Bulut M, Koksal O, et al. (2008) MR
imaging in the detection of diffuse axonal injury with mild traumatic brain
injury. Neurol Res 30: 974–978.
20. McKee AC, Cantu RC, Nowinski CJ, Hedley-Whyte ET, Gavett BE, et al.
(2009) Chronic traumatic encephalopathy in athletes: progressive tauopathy
after repetitive head injury. J Neuropathol Exp Neurol 68: 709–735.
21. Rosengren LE, Karlsson JE, Karlsson JO, Persson LI, Wikkelso C (1996)
Patients with amyotrophic lateral sclerosis and other neurodegenerative diseases
have increased levels of neurofilament protein in CSF. J Neurochem 67:
22. Franz G, Beer R, Kampfl A, Engelhardt K, Schmutzhard E, et al. (2003)
Amyloid beta 1–42 and tau in cerebrospinal fluid after severe traumatic brain
injury. Neurology 60: 1457–1461.
23. Kay AD, Petzold A, Kerr M, Keir G, Thompson E, et al. (2003) Alterations in
cerebrospinal fluid apolipoprotein E and amyloid beta-protein after traumatic
brain injury. J Neurotrauma 20: 943–952.
24. Blennow K, Hampel H, Weiner M, Zetterberg H (2010) Cerebrospinal fluid and
plasma biomarkers in Alzheimer disease. Nat Rev Neurol 6: 131–144.
25. Pelsers MM, Hanhoff T, Van der Voort D, Arts B, Peters M, et al. (2004) Brain-
and heart-type fatty acid-binding proteins in the brain: tissue distribution and
clinical utility. Clin Chem 50: 1568–1575.
26. Honda M, Tsuruta R, Kaneko T, Kasaoka S, Yagi T, et al. (2010) Serum glial
fibrillary acidic protein is a highly specific biomarker for traumatic brain injury
in humans compared with S-100B and neuron-specific enolase. J Trauma 69:
27. Savola O, Pyhtinen J, Leino TK, Siitonen S, Niemela O, et al. (2004) Effects of
head and extracranial injuries on serum protein S100B levels in trauma patients.
J Trauma 56: 1229–1234; discussion 1234.
28. Petzold A, Green AJ, Keir G, Fairley S, Kitchen N, et al. (2002) Role of serum
S100B as an early predictor of high intracranial pressure and mortality in brain
injury: a pilot study. Crit Care Med 30: 2705–2710.
29. Jordan BD (1996) Acute and chronic brain injury in United States National
Team Soccer Players. Am J Sports Med 24: 704–705.
30. Rosengren LE, Wikkelso C, Hagberg L (1994) A sensitive ELISA for glial
fibrillary acidic protein: application in CSF of adults. J Neurosci Methods 51:
31. Olsson A, Vanderstichele H, Andreasen N, De Meyer G, Wallin A, et al. (2005)
Simultaneous measurement of beta-amyloid(1–42), total tau, and phosphorylat-
ed tau (Thr181) in cerebrospinal fluid by the xMAP technology. Clin Chem 51:
32. Raftery AE, Madigan D, Hoeting JA (1997) Bayesian Model Averaging for
Linear Regression Models. Journal of the American Statistical Association. pp
33. Hamberger A, Huang YL, Zhu H, Bao F, Ding M, et al. (2003) Redistribution of
neurofilaments and accumulation of beta-amyloid protein after brain injury by
rotational acceleration of the head. J Neurotrauma 20: 169–178.
34. Zetterberg H, Hietala MA, Jonsson M, Andreasen N, Styrud E, et al. (2006)
Neurochemical aftermath of amateur boxing. Arch Neurol 63: 1277–1280.
35. Donato R, Sorci G, Riuzzi F, Arcuri C, Bianchi R, et al. (2009) S100B’s double
life: intracellular regulator and extracellular signal. Biochimica et biophysica
acta 1793: 1008–1022.
36. Michetti F, Corvino V, Geloso MC, Lattanzi W, Bernardini C, et al. (2011) The
S100B protein in biological fluids: more than a lifelong biomarker of brain
distress. Journal of neurochemistry 120(5): 644–659. doi: 10.1111/j.1471-
37. Hayakata T, Shiozaki T, Tasaki O, Ikegawa H, Inoue Y, et al. (2004) Changes
in CSF S100B and cytokine concentrations in early-phase severe traumatic brain
injury. Shock 22: 102–107.
38. Hasselblatt M, Mooren FC, von Ahsen N, Keyvani K, Fromme A, et al. (2004)
Serum S100beta increases in marathon runners reflect extracranial release
rather than glial damage. Neurology 62: 1634–1636.
39. Kleindienst A, Ross Bullock M (2006) A critical analysis of the role of the
neurotrophic protein S100B in acute brain injury. Journal of neurotrauma 23:
40. Brody DL, Magnoni S, Schwetye KE, Spinner ML, Esparza TJ, et al. (2008)
Amyloid-beta dynamics correlate with neurological status in the injured human
brain. Science 321: 1221–1224.
41. Zetterberg H, Jonsson M, Rasulzada A, Popa C, Styrud E, et al. (2007) No
neurochemical evidence for brain injury caused by heading in soccer. Br J Sports
Med 41: 574–577.
CSF-Biomarkers in Olympic Boxing
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