JOURNAL OF NEUROTRAUMA
Volume 23, Number 8, 2006
© Mary Ann Liebert, Inc.
A Critical Analysis of the Role of the Neurotrophic Protein
S100B in Acute Brain Injury
ANDREA KLEINDIENST1and M. ROSS BULLOCK2
We provide a critical analysis of the relevance of S100B in acute brain injury emphazising the ben-
eficial effect of its biological properties. S100B is a calcium-binding protein, primarily produced by
glial cells, and exerts auto- and paracrine functions. Numerous reports indicate, that S100B is re-
leased after brain insults and serum levels are positively correlated with the degree of injury and
negatively correlated with outcome. However, new data suggest that the currently held view, that
serum measurement of S100B is a valid “biomarker” of brain damage in traumatic brain injury
(TBI), does not acknowlege the multifaceted release pattern and effect of the blood-brain barrier
disruption upon S100B levels in serum. In fact, serum and brain S100B levels are poorly correlated,
with serum levels dependent primarily on the integrity of the blood–brain barrier, and not the level
of S100B in the brain. The time profile of S100B release following experimental TBI, both in vitro
and in vivo, suggests a role of S100B in delayed reparative processes. Further, recent findings pro-
vide evidence, that S100B may decrease neuronal injury and/or contribute to repair following TBI.
Hence, S100B, far from being a negative determinant of outcome, as suggested previously in the hu-
man TBI and ischemia literature, is of potential therapeutic value that could improve outcome in
patients who sustain various forms of acute brain damage.
Key words: cell culture; neurogenesis; prognosis; rat; review; S100B protein; traumatic brain injury
1Department of Neurosurgery, Georg August University, Göttingen, Germany.
2Department of Neurosurgery, Virginia Commonwealth University Medical Center, Richmond, Virginia.
numerous studies have reported a positive correlation
of S100B serum levels with a poor outcome following
traumatic brain injury (TBI), recent findings raise doubts
about the currently held view, that serum measure-
HIS ANALYSIS PROVIDES a critical overview of the
relevance of S100B in acute brain injury. Although
ment of S100B is a valid biochemical marker of brain
In 1965, the first neurotrophic factor was purified from
bovine brain (Moore, 1965), and in 1978 this factor was
demonstrated to consist of two distinct proteins, S100?
and S100? (Isobe et al., 1978). After the identification
of the chromosomal localization of S100 proteins in 1995,
the nomenclature was changed from S100? to S100B and
from S100? to S100A1(Schafer et al., 1995). In the fol-
lowing decade, experimental research was focused on
identifying the specific role of S100B while in the clin-
ical setting the measurement of S100B became more fre-
ROLE OF S100B AS A BIOCHEMICAL
MARKER FOR SEVERITY
OF BRAIN DAMAGE
The early assessment of the injury severity and the con-
sequent prognosis are of major concern for physicians
treating patients suffering from TBI, yet no direct indi-
cator exists to accurately determine the extend of the
brain damage. The measurement of putative biochemical
markers, such as the S100B protein, has been proposed
in this role. In 2003, a thorough review of the role of
S100B as a marker of brain damage was published sum-
marizing the results of 18 clinical studies in a total of
1085 patients (Rothermundt et al., 2003). In 2004 and
2005, another six studies comprising of more than 600
adult patients were performed supporting the correlation
of elevated serum levels of S100B with a poor outcome
after brain injury (Berger et al., 2005; Chen and Zhu,
2005; Pelinka et al., 2004; Savola et al., 2004; Vos et al.,
2004; Wunderlich et al., 2004). The time profile of S100B
reported in the serum was variable, and has been found
to be high directly after TBI, but to normalize within 24
hours after injury in a high percentage of cases, even in
those with a bad outcome (Jackson et al., 2000). Another
study in head injured patients demonstrated a delayed in-
crease of S100B serum levels on the 6thday after injury
with a correlation to outcome and the authors attributed
these delayed increases to the development of secondary
brain cell damage (Raabe and Seifert, 2000). After con-
trolled cortical impact injury in the rat, S100B serum lev-
els were elevated up to 48 hours post-injury (Rothoerl et
In view of this substantial body of evidence demon-
strating an association between S100B and bad outcome
after TBI, it is important to be aware that proof of asso-
ciation is not proof of causation, in science, and this is
especially true of S100B. Nevertheless, strong evidence
exists that S100B serum levels are increased without con-
comitant brain damage (Anderson et al., 2001a,b; Ashraf
et al., 1999; Kleine et al., 2003; Nygren De Boussard et
al., 2004; Pelinka et al., 2003; Svenmarker et al., 2002).
The extracerebral origin of S100B has also been recog-
nized in several studies (Arcuri et al., 2002; Donato,
1991; Suzuki et al., 1984; Zimmer and Van Eldik, 1987)
and this data conflicts with the interpretation of S100B
serum levels following TBI. Moreover, this data suggests
that elevated S100B serum levels after TBI or other brain
insults (Donato, 1976) are simply due to an increased pas-
sage of cerebral S100B from the central nervous system
via a damaged blood-brain-barrier. Thus, increased serum
levels may not be truly reflective of increased astrocytic
S100B release or production in response to trauma. Al-
though S100B measurements in the cerebrospinal fluid
of patients following brain insults allow a more accurate
assessment of the true cerebral S100B levels (Hayakata
et al., 2004; Kleine et al., 2003; Petzold et al., 2003; Shore
et al., 2004), the exact function and effects of increased
cerebral levels of S100B after TBI are not thoroughly un-
OF THE S100B PROTEIN
The S100B protein belongs to a multigenic family of
low molecular weight (9-13 kD) calcium-binding S100
proteins (Donato, 2001; Heizmann et al., 2002). S100B
is most abundant in glial cells of the central nervous sys-
tem, predominately in astrocytes (Donato, 1986), and
constitutes 1–1.5 ?g/mg of soluble protein (Matsutani et
al., 1985). S100 proteins contain no detectable carbohy-
drate, lipid, nucleic acid or phosphate, and are dimers
consisting of two types of subunits of either identical or
different amino acid composition (Fig. 1). S100 proteins
are highly conserved in aminoacid composition among
vertebrate species (Donato, 1986), and comparison of the
human and bovine S100A1and the human and rat S100B
subunit reveals an almost complete homology (Isobe et
Intracellularly, S100B is involved in signal transduc-
tion via the inhibition of protein phoshorylation, regula-
tion of enzyme activity and by affecting the calcium
homeostasis (Rustandi et al., 1998; Wilder et al., 1998;
Zimmer and Van Eldik, 1986). Moreover, S100B is func-
tionally involved in the regulation of cell morphology by
interaction with elements of the cytoplasmatic cy-
toskeleton. S100B is actively secreted from astroglia via
an unknown mechanism, and also exerts extracellular
functions (Fig. 2). In cultured rat astrocytes, S100B re-
lease can be stimulated by 5 HT 1a agonists (Shashoua
et al., 1984; Van Eldik and Zimmer, 1987; Whitaker-
Azmitia et al., 1990) and is observed within a few min-
utes after activation of A1 adenosine or mGlu3
metabotropic glutamate receptors and release may last up
to 10 hours (Ciccarelli et al., 1999).
Initial studies have shown, that S100B stimulates neu-
rite outgrowth and enhances cell maintenance in cultures
KLEINDIENST AND ROSS BULLOCK
of embryonic chick cerebral cortex neurons (Kligman
and Marshak, 1985; Winningham-Major et al., 1989),
and stimulates proliferation of primary rat astrocytes
(Selinfreund et al., 1991). Further investigations also
confirmed a dose-dependent action of S100B exerting a
neuroprotective and neurotrophic influence at nanomo-
lar concentrations (Huttunen et al., 2000; Li et al., 1998;
Rickmann et al., 1995), but at micromolar concentra-
tions, an activation of the inducible nitric oxide (NO)
synthase was seen with subsequent NO generation, po-
tentially leading to astrocytic death (Hu et al., 1997;
Petrova et al., 2000).
Since the S100B gene is localized on chromosome 21
(Schafer et al., 1995) and elevated S100B levels have
been demonstrated in Down’s syndrome and Alzheimer’s
disease (Griffin et al., 1989), S100B has been suggested
to be involved in the development of these brain patholo-
gies. Furthermore, S100B expression correlates with neu-
ritic plaque density (Sheng et al., 1994), with the density
of dystrophic neurites over expressing ?-amyloid pre-
cursor protein (Mrak et al., 1996), and S100B induced
the expression of the pro-inflammatory interleukin-1
(Griffin et al., 1989; Liu et al., 2005; Sheng et al., 1994).
Whether the increase of S100B in neurodegenerative dis-
eases is part of a compensatory response, contributes to
the pathology or results from dysregulated feedback
loops, has not been clarified yet.
NEUROPROTECTIVE EFFECTS OF S100B
After release into the extracellular fluid, S100B acts in
an autocrine and paracrine manner. In vitro studies
demonstrate mitogenic properties of S100B, such as an
increased proliferative activity on melanoma cell lines at
concentrations below 50 nM and above 5 ?M (Klein et
al., 1989), as well as on rat C6 glioma cells at 50 pM to
1.5 nM (Selinfreund et al., 1991), preferably in its oxi-
dized state (Scotto et al., 1998). Stimulation of mitogen-
activated protein kinase (ERK) by S100B provides evi-
dence for a proliferative effect on primary neonatal rat
astrocytes (Goncalves et al., 2000).
Different studies provide evidence for neurotrophic
properties of S100B. In cultured mesencephalic rat neu-
rons (Azmitia et al., 1990) and dorsal root ganglia (Van
Eldik et al., 1991), S100B has been shown to promote
neurite outgrowth (Kligman and Marshak, 1985; Win-
ningham-Major et al., 1989). In primary rat spinal cord
culture, S100B rapidly promoted reassembly and/or sta-
S100B IN ACUTE BRAIN INJURY
spectively, I?–IV?. Binding of Ca2?to each S100B monomer causes a reorientation of helix III relative to all other helices with
consequent reorientation of the hinge region (H). These changes result in the exposure to the solvent of a surface defined by
residues (magenta). (Reproduced, with permission, from Donato, 2001.)
Structure of the S100B protein. One S100B monomer is yellow, and the other one blue; helices are indicated I–IV, re-
bilization of the cytoskeletal system (Nishi et al., 1997),
and prevented apoptosis in a neuroblastoma clonal cell
line (Brewton et al., 2001).
In vivo, astrocytosis and neurite proliferation occurred
in transgenic mice overexpressing S100B as demon-
strated by increased immunoreactivity to glial fibrillary
acid protein (GFAP) and an extensive branching of glial
processes as well as by an increased immunoreactivity to
the neuronal marker ?III-tubulin within the hippocampus
while the number of hippocampal neurons remained un-
changed (Reeves et al., 1994). More importantly, how-
ever, after sciatic nerve section in newborn rats, S100B
rescued motor neuron death and preserved neuron diam-
eter (Iwasaki et al., 1997).
The trophic effects are thought to be mediated, at least
in part, by receptors of advanced glycation end products
(RAGE) inducing neurite outgrowth, activating the tran-
scription factor NF-?B, and promoting expression of the
anti-apoptotic protein Bcl-2 (Huttunen et al., 2000; Li
et al., 1998; Rickmann et al., 1995). In vitro, S100B has
been shown to inhibit proteinkinase C (PKC)–dependent
phosphorylation of p53, thereby providing evidence for
an involvement in cell-cycle processes at the G0-G1/S
transition (Rustandi et al., 1998; Wilder et al., 1998; Zim-
mer and Van Eldik, 1986).
In addition to its trophic properties, a neuroprotective
effect of S100B was found in vitro.S100B decreased neu-
ronal cell death and mitochondrial dysfunction in rat hip-
pocampal neurons after glucose deprivation, and in-
creased intracellular free calcium (Barger et al., 1995).
S100B is, thus, thought to be involved in regulation of
energy metabolism by stimulation of the fructose-1,6-
biphosphate aldolase (Zimmer and Van Eldik, 1986) and
phosphoglucomutase (Landar et al., 1996). S100B has
also been implicated in cytosolic Ca2?buffering (Xiong
et al., 2000). In cultures of embryonal chick and neona-
tal rat neurons, S100B was found to protect against glu-
tamate- and staurosporin-induced damage (Ahlemeyer et
al., 2000). The above-mentioned neuroprotective and
neurotrophic properties of S100B have not been eluci-
dated after TBI.
S100B AND COGNITIVE FUNCTION
Besides this experimental evidence for the beneficial
effects of S100B on neuronal maintenance, a specific
role of S100B has also been proposed at a higher level
in developmental plasticity (Marshak, 1990), and in cell
processes thought to be involved in learning and mem-
ory, such as long-term potentiation (Fazeli et al., 1990).
This has been confirmed by injection of S100B anti-
serum into the hemisphere of chicks causing amnesia for
a passive avoidance task (O’Dowd et al., 1997). Vice
versa, S100B infused into the rat hippocampus has been
shown to facilitate long-term memory for an inhibitory
avoidance task (Mello e Souza et al., 2000). Finally,
light- and electron microscopic studies are consistent
with the hypothesis, that S100B plays a role in lesion-
induced collateral sprouting and reactive synaptogenesis
(Huttunen et al., 2000; Li et al., 1998; McAdory et al.,
1998; Rickmann et al., 1995), and that repair may occur
by interaction with growth factors (Gomide and Chadi,
Cognitive impairment is one of the most disabling fea-
tures of TBI, and different studies have demonstrated an
increased susceptibility of the hippocampus to injury, the
brain region most critical for cognitive performance,
specifically learning and memory (Hicks et al., 1993;
McIntosh et al., 1989; Miller et al., 1990; Miyazaki et
al., 1992; Povlishock, 1993). Recent findings of active
neuroprogenitor cells within the walls of the lateral ven-
tricle and the sub-granular zone of the dentate gyrus of
the hippocampus in adult mammals provide evidence of
an intrinsic regenerative potential in the brain (Kuhn et
al., 1996; Lois and Alvarez-Buylla, 1993; Reynolds and
Weiss, 1992). Thus, the increased cellular proliferation
and subsequent neuron formation within the hippocam-
pus following TBI (Chirumamilla et al., 2002; Dash et
al., 2001) may be an important innate repair mechanism
of the brain. In addition to the growth factors that have
been found to up-regulate neurogenesis (Yoshimura et
al., 2001), the neurotrophic protein S100B is also a po-
tential contributor to hippocampal network repair after
TBI, as shown by its promotion of memory consolida-
tion in the normal rat during development (Mello e Souza
et al., 2000).
However, in contrast, transgenic mice produced by an
insertion of the human S100B gene on chromosome 21,
demonstrate an overexpression of S100B throughout the
brain and show behavioral abnormalities. Assessment of
cognitive function with a wide range of behavioral tests
revealed hippocampal-dependent deficits although the
impaired performance of S100B + mice may also reflect
the involvement of non-hippocampal areas (Winocur et
al., 2001). Studies in these S100B transgenic mice at dif-
ferent ages suggest, that S100B may accelerate hip-
pocampal development as demonstrated by an increased
density of hippocampal dendrites at the earliest stages
followed by an enhanced aging and loss of dendrites
(Shapiro et al., 2004; Whitaker-Azmitia et al., 1997).
These findings are supported by an accumulation of
S100B found during developmental synaptogenesis in
normal rodents (Tramontina et al., 2002) and in humans
(Portela et al., 2002).
KLEINDIENST AND ROSS BULLOCK
EFFECT OF S100B ON NEURONAL
FUNCTION FOLLOWING IN VITRO
TRAUMATIC BRAIN INJURY
Because of the complexity of studying TBI in vivo, a
reductionist tissue culture approach has the advantage of
studying the effect of certain substances on brain cells
following injury specifically by reducing potential inter-
actions. One in vitro approach to mimic TBI, induces a
50-millisecond cell strain (stretch), known to be a major
component of in vivo TBI (Thibault et al., 1992), on neu-
ronal plus glial cell cultures grown on deformable sili-
cone membranes (Ellis et al., 1995). Since S100B is
found in glial conditioned media, often used to promote
neuronal survival in culture, and acts long term, the ef-
fect of S100B on normal neuronal membrane integrity,
its release from strain (stretch)-injured neuronal plus glial
cultures, and the capacity of S100B to affect delayed neu-
S100B IN ACUTE BRAIN INJURY
ulation of a large variety of cell activities, as indicated. S100B has been shown to be released into the extracellular space, albeit
with mechanisms that remain to be elucidated, and exerts effects on other cells. S100B may have a dual role, like a Janus face.
Beside beneficial neurotrophic activities, S00B can stimulate astrocytes and microglia to produce pro-inflammatory cytokine at
supraphysiological concentrations. (Modified, with permission, from Donato, 2001, and Van Eldik and Wainwright, 2003.)
Summary of intracellular and extracellular functional roles of S100B. Within cells, S100B may participate in the reg-
ronal injury were, therefore, determined following in
vitro TBI (Willoughby et al., 2004).
In normal un-injured neuronal plus glial cultures, the
level of S100B in the growth medium is measurable (Fig.
3). Fifteen seconds after injury, there was a dramatic rise
in S100B, indicating cell release of preformed S100B, and
a continued release of S100B until at least 48 hours post-
injury (Slemmer et al., 2002; Willoughby et al., 2004). The
injury-induced S100B release is likely to be the result of
at least two possible processes. Firstly, electron micro-
scopic studies of stretch-injured astrocyte cultures show,
that immediately after stretch-injury structural and mem-
brane integrity is severely compromised (Ellis et al., 1995).
Secondly, since an injury-induced ATP and glutamate re-
lease has been shown also (Ahmed et al., 1998, 2002), the
release of S100B might be in part due to an astrocyte re-
ceptor activation by ATP and glutamate (Ciccarelli et al.,
1999). The finding of an increased S100B release at 24 and
48 hours post-injury may, thus, imply that this is a longer-
term metabolic response to brain damage, which promotes
a “healing process” in injured neurons or astrocytes.
The delayed neuronal injury observed in this stretch-
injury TBI model has not been pharmacologically pre-
ventable, except when agents were given before or sec-
onds to minutes after injury (Chen et al., 2003). The
delayed neuronal injury is in large part initiated by
NMDA receptor activation, occurring immediately after
trauma and resulting in an alteration in GABA and
AMPA synaptic function as well as in an injury-induced
delayed depolarization (Goforth et al., 1999; Kao et al.,
2004; Tavalin et al., 1995, 1997). In this stretch-injury
model, even when the S100B administration was post-
poned to 24 hours after injury, the delayed neuronal in-
jury at 48 hours was still reduced by S100B (Fig. 4). A
possible mechanism by which S100B may reduce the de-
layed neuronal injury is by affecting the compromised
bioenergetics in injured cells (Barger et al., 1995). Neu-
ronal mitochondrial function in neuronal plus glial cul-
tures is suppressed at 15 minutes after stretch-injury, but
recovers to normal by 24 hours. However, in injured pure
neuronal cultures, the mitochondrial membrane potential
does not recover (Ahmed et al., 1998). This suggests, that
astrocytes, and potentially astrocyte produced S100B,
may have an important bioenergetic reparative effect.
ASSESSMENT OF CEREBRAL S100B
LEVELS BY MAGNETIC RESONANCE
While in cell cultures the S100B release continues to
increase up to 48 hours following injury (Slemmer et al.,
2002; Willoughby et al., 2004), S100B serum levels in
patients are normally highest directly after injury, and be-
come normalized within 24 hours in a high percentage
of cases, even in those patients with a bad outcome (Jack-
son et al., 2000). The underlying mechanism of the pas-
sage of S100B through the blood–brain barrier (BBB) has
not been clarified yet, nor do data exist about cerebral
S100B levels and their correlation to serum S100B lev-
els, in humans. Since one of the purposes of the BBB is
to prevent proteins to enter the brain, there is reasonable
KLEINDIENST AND ROSS BULLOCK
ronal plus glial cultures. S100B release occurred immediately
after injury (7.5-mm membrane displacement) and increased
with time. Values are means ? SEM. The SEM for no injury
is too small to display graphically. N ? 6–8 wells; a, p ? 0.05
versus no injury; b, p ? 0.05 versus 15 sec. (Modified, with
permission, from Willoughby et al., 2004.)
Release of S100B after severe stretch injury of neu-
S100B on delayed neuronal injury in stretch-injured neuronal
plus glial cultures. S100B, when given at 24 h after injury, de-
creased delayed neuronal injury by one-half as indicated by pro-
pidium iodide uptake at 48 h post-injury. Values are means ?
SEM. N ? 6; a, p ? 0.05 versus uninjured control; b, p ? 0.05
versus injury without S100B. (Modified, with permission, from
Willoughby et al., 2004.)
The effect of delayed administration of 10 or 100 nM
doubt whether S100B of cerebral origin may be able to
cross the intact BBB in the opposite direction. Measure-
ments of S100B cerebrospinal fluid levels allow a true
assessment of the cerebral S100B release following brain
insults (Hayakata et al., 2004; Kleine et al., 2003; Pet-
zold et al., 2003; Shore et al., 2004), but require for repet-
itive measurements either lumbar or ventricular cere-
brospinal fluid drainage.
The principle of spectroscopy is widely applied in
chemistry for the analysis of molecules in solution, and
is a powerful tool for determining the structure of bio-
logical macromolecules. Similarly, MR spectroscopy can
be used to identify important molecules in living tissue.
Protons often are used for MR spectroscopy because of
their high natural abundance and high nuclear magnetic
sensitivity. Despite the huge number of biomolecules in
tissue, relatively few are identifiable in vivo because only
freely mobile compounds that are present in substantial
concentrations give enough signals to be detected. The
concentrations of metabolites of interest are in the mil-
limolar range; water protons are a thousand times as com-
mon. For this reason, water resonance has to be sup-
pressed so that the other molecules can be detected.
Recently, MR proton spectroscopy has been shown to
identify brain metabolites like N-acteylaspartate (NAA),
creatine, choline and lactate (Bruhn et al., 1989; Frahm
et al., 1989a,b,c). Moreover, we demonstrated MR pro-
ton spectroscopy to detect different concentrations of
aqueous solutions of S100B in vitro, with a strong cor-
relation between the S100B concentration and the area
under the curve of the respective S100B MR peak at 4.5
ppm (Kleindienst et al., 2005b).
First, in vivo studies in a TBI model examined the ca-
pability of MR proton spectroscopy to assess cerebral
S100B levels, and then compared them with the corre-
sponding serum S100B concentration (Kleindienst et al.,
2005b). Second, in normal non-injured animals, in which
the BBB was intact, cerebral S100B levels were exoge-
nously increased by an intraventricular S100B infusion,
and cerebral levels were then measured by MR proton
spectroscopy. When the serum concentration of S100B
was measured, it did not increase according to the notion
that proteins do not cross an intact BBB (Fig. 5).
Furthermore, following experimental TBI, cerebral
S100B levels as detected by MR proton spectroscopy (Fig.
6) did not correlate with the serum S100B levels measured
at corresponding time-points (Kleindienst et al., 2005b).
The serum S100B levels increased immediately after in-
jury, peaked at 3 hours, and returned to normal values at
24 hours after injury (Fig. 7). This time profile of serum
S100B levels parallels the BBB disruption found after ex-
perimental TBI (Povlishock et al., 1978). In contrast, cere-
bral S100B levels as quantified by MR proton spectroscopy
were raised slightly 3 hours after injury, and increased sig-
nificantly thereafter to more than twice-these values by day
5. Thus, no clear relationship exists between the cerebral
S100B dynamics and the serum S100B levels.
These findings thus suggest that the release of cerebral
S100B into blood is modulated by additional factors like
the integrity of the BBB, cerebral blood flow, or possi-
ble degradation, synthesis, and sequestration by other or-
gans than the brain. The very rapid occurrence of high
serum S100B levels found immediately after injury may
represent the contribution of intracellular S100B origi-
nating from damaged astrocytes and passing through a
“widely open” blood-brain-barrier for a limited time, as
shown by BBB tracer studies. If the BBB is closed, even
the astrocytic synthesis, that has been clearly shown, may
not be reflected in serum levels. The assessment of cere-
bral S100B levels by MR proton spectroscopy may thus
offer an easily accessible tool to non-invasively monitor
endogenous repair mechanisms essential for cognitive re-
covery in patients after brain injuries.
EFFECT OF S100B ON COGNITIVE
While in vitro studies confirm a trophic effect of
S100B on neuronal survival and neurite outgrowth, the
S100B IN ACUTE BRAIN INJURY
ventricular S100B infusion. Measurement of serum S100B lev-
els in serum (ng/mL; means ? SEM; the standard errors are too
small to display graphically) and the area under the curve (AUC)
of the S100B peak in MR spectroscopy (means ? SEM) at dif-
ferent time points following an intraventricular S100B infusion
for 5 days. a, p ? 0.05 versus 30 min; MRS, magnetic reso-
nance proton spectroscopy. (Modified, with permission, from
Kleindienst et al., 2005b.)
Serum and cerebral levels of S100B after an intra-
importance of S100B after the pathological event of TBI
has not been clarified. Whenever the S100B production
is challenged excessively and supra-physiologically, as
in TBI, the capability of astrocytes to produce S100B
may not equal the raised demand for S100B. This hy-
pothesis has been examined by an exogenous adminis-
tration of S100B into the lateral ventricle following fluid
percussion injury in the rat (Kleindienst et al., 2004).
Cognitive recovery was assessed by the Morris water
maze 5 weeks post-injury and revealed a significantly
improved performance following intraventricular S100B
infusion (Kleindienst et al., 2004). This beneficial effect
of S100B in vivo occurred at nanomolar concentrations
and is in agreement with in vitro experiments (Barger
et al., 1995; Huttunen et al., 2000), while micromolar
concentrations induced apoptosis in vitro (Li et al.,
1998). Interestingly, the S100B concentration used in
this in vivo TBI study is around 100 times higher than
levels in normal controls (?0.2 ng/mL, ?0.8 ng/mL in
older individuals), and 10 times higher than S100B lev-
els found in acute brain pathologies (Drohat et al., 1998;
Green et al., 1997; Peskind et al., 2001; Pleines et al.,
The importance of S100B in the acutely injured brain
is thus not well known yet. After electroconvulsive ther-
apy in patients, increased serum S100B levels were as-
sociated with an improved cognitive performance
(Agelink et al., 2001). Seizures were found to induce as-
trocyte activation, followed by the release of neurotrophic
factors which contribute to synaptic remodeling (Kragh
KLEINDIENST AND ROSS BULLOCK
fluid percussion injury, the T2-weighted image did not show any relevant edema or other intracranial pathology. The white box
indicates the region of interest (RoI) from which the subsequent spectrum was acquired (A). In MR proton spectroscopy, a S100B
specific peak at 4.5 ppm close to the water peak can be depicted, which is not found in control animals (spectra of the injury
side; B). (Modified, with permission, from Kleindienst et al., 2005b.)
Magnetic resonance (MR) imaging and proton spectroscopy after lateral fluid percussion injury. On day 5 after lateral
percussion injury. Measurement of serum S100B levels in serum
(ng/mL; means ? SEM) and the area under the curve (AUC) of
the S100B peak in magnetic resonance (MR) spectroscopy
(means ? SEM) at different time points in animals subjected to
lateral fluid percussion injury. a, p ? 0.05 versus uninjured con-
trols. MRS, magnetic resonance proton spectroscopy. (Modified,
with permission, from Kleindienst et al., 2005b.)
Serum and cerebral levels of S100B after lateral fluid
et al., 1993). Both, an endogenous injury-induced release
of S100B and the additional exogenous intraventricular
administration of S100B, may thus contribute to the re-
covery of cognitive performance following experimental
TBI. This beneficial effect of S100B on functional re-
covery can be mediated by the neurotrophic properties
demonstrated in vitro (Kligman and Marshak, 1985;
Selinfreund et al., 1991). Alternatively, it is also possi-
ble, that the known neuroprotective effects of S100B on
mitochondrial function (Barger et al., 1995) could cause
increased neuronal survival and thus improved outcome
following experimental TBI.
S100B IN ACUTE BRAIN INJURY
ution of bromodeoxyuridine (BrdU)–immunoreactive cells within the ipsilateral dentate gyrus at different time points following
lateral fluid percussion injury. (A) Higher magnification showing that BrdU-immunoreactive cells on day 5 post-injury are pre-
dominately identified in the area of their origin, the subgranular zone. The arrow points to a typical cluster of cells. (B) Lower
magnification of the ipsilateral dentate gyrus 5 weeks post-injury. Some BrdU-immunoreactive cells are now located in the gran-
ular cell layer (arrows), suggesting their migration from the subgranular zone. (Modified, with permission, from Kleindienst et
Assessment of injury-induced progenitor cell proliferation in the hippocampus. Photomicrographs showing the distrib-
traumatic brain injury (TBI). Confocal photomicrographs show the co-localization of the neuronal marker NeuN and the glial
marker glial fibrillary acidic protein (GFAP) with bromodeoxyuridine (BrdU) immunoreactivity in the dentate gyrus at 5 weeks
post-injury. (A–C) NeuN (red), BrdU (green) and co-localization of NeuN and BrdU (orange). (D–F) GFAP (red), BrdU (green)
and co-localization of GFAP and BrdU (orange). SGZ, subgranular zone. (Modified, with permission, from Kleindienst et al.,
Identification and quantification of the maturational fate of progenitor cells in the ipsilateral dentate gyrus following
EFFECT OF S100B ON HIPPOCAMPAL
While S100B has been found to exert mitogenic prop-
erties (Goncalves et al., 2000; Klein et al., 1989; McAdory
et al., 1998; Rustandi et al., 1998; Selinfreund et al., 1991;
Wilder et al., 1998), and has been implicated in develop-
mental plasticity (Marshak, 1990), lesion-induced reactive
synaptogenesis (Fazeli et al., 1990) and found to facilitate
learning and memory (Mello e Souza et al., 2000; O’Dowd
et al., 1997), the effect of S100B on cellular responses ini-
tiated following TBI has not yet been determined.
The hippocampus is a region critical for learning and
memory. It displays increased susceptibility to injury, and
cognitive impairment following injury has been linked to
hippocampal dysfunction (Hicks et al., 1993). Recent
findings of neurogenesis within the subgranular zone of
the dentate gyrus of the hippocampus in adult mammals
provides a possible source to replace lost neurons (Eriks-
son et al., 1998; Gould and Gross, 2002; Kornack and
Rakic, 1999). Indeed, neuronal progenitor cells within the
hippocampus present increased cellular proliferation and
subsequent neuron formation following experimental
TBI (Chirumamilla et al., 2002; Dash et al., 2001). This
adult neurogenesis has been found to be actively regu-
lated by hippocampal astrocytes, which are a cellular
source for mitogenic/neurotrophic factors and thus pro-
mote proliferation of adult neural stem cells and instruct
the fate commitment of developing neurons (Song et al.,
2002). Consequently, the astrocytic neurotrophic protein
S100B is a potential candidate to increase this progeni-
tor cell proliferation and subsequent neuron formation
following TBI, thereby enhancing hippocampal network
repair and improving cognitive recovery.
The progenitor cell proliferation in the dentate gyrus
of the hippocampus has been assessed by injecting the
mitotic marker bromodeoxyuridine (BrdU) on day 2 post-
injury following lateral fluid percussion injury, and
intraventricular S100B administration in the rat (Klein-
dienst et al., 2005a). The typical lesion-induced prolifer-
ative response in the ipsi-lateral dentate gyrus was sig-
nificantly increased by more than 40% by intraventricular
S100B infusion on day 5 post-injury (Fig. 8A). In vehi-
cle-infused animals, the number of BrdU-immunoreac-
tive cells returned to control values by 5 weeks post-in-
jury, while in S100B infused animals the survival of
progenitor cells was still significantly increased 5 weeks
post-injury (Fig. 8B). Although proliferative effects of
S100B have been found in vitro in cultures of different
cell lines (Goncalves et al., 2000; Klein et al., 1989;
Selinfreund et al., 1991), in the non-injured control ani-
mals no relevant mitogenic effect of S100B was evident,
while in injured animals the progenitor cell proliferation
was almost doubled (Kleindienst et al., 2005a). Hence,
S100B may act in concert with other factors released fol-
lowing TBI thereby contributing to a stimulated endoge-
nous proliferative response.
The differentiational fate of the hippocampal progen-
itor cells was quantified by double-labeling with cell-spe-
cific markers to determine, whether the enhanced prolif-
erative response following TBI and an intraventricular
S100B infusion promoted subsequent neurogenesis
(Kleindienst et al., 2005a). BrdU immunoreactive cells
in the dentate gyrus were double-labeled with the neu-
ronal marker NeuN to identify unambiguously newly
generated cells as neurons (Fig. 9). NeuN is a transcrip-
tion factor that is expressed in the nucleus and cytoplasm
of mature neurons (Magavi et al., 2000; Mullen et al.,
1992), and is widely used as a neuron-specific marker.
Following injury, the percentage of progenitor cells,
which differentiated into neurons were significantly en-
hanced by intraventricular S100B infusion, as demon-
strated by an increased number of BrdU-positive cells
also demonstrating NeuN immunoreactivity (Kleindienst
et al., 2005a). The mechanisms that are involved in the
neurogenic effect of S100B are not known yet and ap-
pear to be initiated by an insult since in un-injured con-
trol animals no hippocampal progenitor cell proliferation
was found. However, some of the neurotrophic effects of
S100B may have promoted the survival of the newly gen-
erated neurons. After neuronal disruption, S100B has
been found to rescue motor neurons and preserve neuron
diameter (Iwasaki et al., 1997), to promote re-assembly
and/or stabilization of the cytoskeletal system rapidly
(Nishi et al., 1997), and to prevent apoptosis (Brewton et
Finally, the contribution of the enhanced hippocampal
neurogenesis following experimental TBI and intraven-
tricular S100B infusion to the functional integration of
neurons was determined (Kleindienst et al., 2005a). To
do this, the histological data were correlated with the
functional recovery as assessed by the Morris water maze.
The Morris water maze is a standard method for assess-
ing cognitive function of rats (Brandeis et al., 1989), and
is known to be sensitive to hippocampal damage (Olton
and Werz, 1978; Wolfer and Lipp, 1992). The signifi-
cantly increased neurogenesis following TBI and an in-
traventricular S100B infusion was correlated with an im-
proved cognitive performance (Fig. 10). Hence, these
“newborn neurons” can be assumed to have functionally
integrated into existing neuronal circuits thereby con-
tributing to improved cognitive function. However, the
cellular and synaptic mechanisms of this plasticity in
newly generated neurons are largely unknown, although
KLEINDIENST AND ROSS BULLOCK
it has been suggested, that these neurons may have spe-
cific properties to facilitate learning (Nottebohm, 2002;
Shors et al., 2001). Furthermore, light and electron mi-
croscopic studies are consistent with the hypothesis, that
S100B plays a role in lesion-induced reactive synapto-
genesis (Huttunen et al., 2000; Li et al., 1998; McAdory
et al., 1998; Rickmann et al., 1995).
Collectively, S100B has thus been demonstrated to in-
crease hippocampal stem/progenitor cell proliferation
and neuronal differentiation following TBI, and this en-
hanced neurogenesis is correlated with an improved cog-
nitive recovery (Kleindienst et al., 2005a). These find-
ings stress the importance of astrocytic factors for
neurogenesis and provide compelling evidence for a ther-
apeutic potential of S100B improving functional recov-
ery following TBI. Further studies are needed to eluci-
date the beneficial role of S100B on hippocampal
network repair, thus offering a potential therapy to aug-
ment important innate repair mechanisms of the brain in
order to promote memory consolidation in patients with
Recent findings from our laboratories elucidate a con-
troversial new role of the astrocytic neurotrophic protein
S100B in acute brain injuries. We demonstrated an in-
jury-induced release of S100B from astrocytes cocultured
with neurons with a time-profile that may suggest a par-
ticipation of S100B in delayed reparative processes,
which should be elucidated further. The injury-induced
cerebral S100B release may be assumed to be a combi-
nation of a passive release by damaged astrocytes and an
active release by stimulated astrocytes initiating repair
mechanisms, with both release patterns varying over time
and in the presence of secondary insults.
We found that serum S100B levels do not reflect the
corresponding cerebral S100B levels. The passage of
cerebral S100B is modulated by the BBB on its way into
the extracerebral compartment. Additional contribution
to serum S100B originates from extracerebral sources. In
parallel, S100B is subject to degradation and elimination.
Thus, when interpreting S100B serum levels one has to
keep in mind, that they reflect different factors and de-
pend mostly on the integrity of the BBB rather than re-
flect the cerebral S100B level. We established MR pro-
ton spectroscopy to more accurately reflect cerebral
S100B levels and thus provide a clinical method to non-
invasively monitor whole brain S100B levels repeatedly
in patients with brain insults. The determination of cere-
bral S100B levels may allow a more accurate estimation
of the prognosis in head-injured patients.
In experimental TBI, we confirmed, both in vitro and
in vivo, a beneficial effect of S100B on neuronal main-
tenance, neurogenesis, and cognitive performance,
thereby underlining the importance of astrocytic factors
for neuronal function. By assessing brain stem/progeni-
tor cell proliferation and neuronal differentiation follow-
ing experimental TBI and S100B treatment in vivo, we
found these effects to be driven, at least in part, by the
mitogenic and neurotrophic properties of S100B. Fur-
thermore, we demonstrated S100B to promote the func-
tional integration of injury-induced hippocampal neu-
rogenesis and to enhance cognitive recovery. These
findings offer a possible new treatment approach utiliz-
ing the experimentally proven beneficial effects of S100B
clinically to enhance restoration of cognitive function fol-
lowing acute brain injury in patients. However, the liter-
ature supporting a pathologic role of S100B in neurode-
generative diseases, particularly in Alzheimer’s disease,
has to be acknowledged as well. Whether the contrasting
findings in TBI and neurodegenerative diseases are the
result of an interaction with additional factors modulat-
ing the effect of S100B under different circumstances, or
are due to a variation of the concentration and duration
of exposure to S100B, is not clear yet. Furthermore, in
neurodegenerative diseases, the function of S100B has
not been ultimately clarified, both a causative, detrimen-
tal one, and a compensatory one has to be considered.
S100B IN ACUTE BRAIN INJURY
neurogenesis in the ipsilateral dentate gyrus. The cognitive
function assessed by the Morris water maze 5 weeks post-in-
jury and the hippocampal neurogenesis as quantified by the per-
centage of BrdU-immunoreactive cells co-expressing NeuN,
demonstrated a correlation between an improved cognitive
recovery and the number of newly generated neurons in the
dentate gyrus following TBI and intraventricular S100B admin-
istration (r ? 0.86). (Modified, with permission, from Klein-
dienst et al., 2005a.)
Correlation of cognitive function and injury-induced
Complementary research is warranted to elucidate the
specific role of S100B in acute and chronic brain injury.
Taken together, although the desire for a marker of
brain damage is reasonable, designating S100B for this
role warrants considerable simplification. Moreover, we
propose that S100B, far from being a negative determi-
nant of outcome, as suggested previously in the human
TBI and ischemia literature, may improve neurogenesis
and functional recovery following acute brain injury and
may be in fact potentially a new treatment option, which
might improve the outcome of patients with many forms
of acute brain damage.
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