? The?Journal?of?Clinical?Investigation http://www.jci.org Volume 115 Number 8 August 2005
The hematopoietic factor G-CSF is a neuronal
ligand that counteracts programmed cell
death and drives neurogenesis
Armin Schneider,1 Carola Krüger,1 Tobias Steigleder,2,3,4 Daniela Weber,1 Claudia Pitzer,1
Rico Laage,1 Jaroslaw Aronowski,5 Martin H. Maurer,6 Nikolaus Gassler,7 Walter Mier,8
Martin Hasselblatt,9 Rainer Kollmar,3 Stefan Schwab,3 Clemens Sommer,10 Alfred Bach,1
Hans-Georg Kuhn,4,11 and Wolf-Rüdiger Schäbitz2,3
1Axaron Bioscience AG, Heidelberg, Germany. 2Department of Neurology, University of Münster, Münster, Germany. 3Department of Neurology,
University of Heidelberg, Heidelberg, Germany. 4The Arvid Carlsson Institute for Neuroscience at the Institute for Clinical Neuroscience,
Sahlgrenska Academy, Gothenburg University, Gothenburg, Sweden. 5Department of Neurology, University of Texas — Houston, Houston, Texas, USA.
6Institute for Physiology and Pathophysiology, 7Institute for Pathology, and 8Department of Nuclear Medicine, University of Heidelberg, Heidelberg, Germany.
9Institute for Neuropathology, University of Münster, Münster, Germany. 10Department of Neuropathology, Johannes Gutenberg–University of Mainz,
Mainz, Germany. 11Department of Neurology, University of Regensburg, Regensburg, Germany.
Stroke remains one of the most urgent medical problems of our
times, growing in importance due to the demographic changes
in industrialized societies. Treatment with tissue-plasmino-
gen activator is limited by side effects and by the fact that it
must be initiated within a short window of time, so that only
a small percentage of all stroke patients undergo thrombolysis
(1). Numerous neuroprotective strategies aiming at important
mechanisms such as glutamate toxicity or free radical formation
have failed due to lack of efficacy or intolerable side effects. It is
therefore believed that a successful treatment strategy should be
well tolerated, not interfere with essential brain physiology, and
approach several pathophysiological mechanisms in parallel. In
addition to effects on acute infarct evolution, novel strategies
should also impact long-term functional outcome. Recovery of
specific functions and improvement of activities of daily living
are caused by intrinsic changes in existing neurons or networks
or by the generation of new neurons from progenitor cells.
Regarding the latter, enhancement of neurogenesis in the post-
ischemic brain now appears to be an attractive strategy. Neural
progenitor cells residing in the adult brain can indeed initiate
a compensatory response to ischemic events that results in the
production of new neurons (2, 3).
G-CSF is a 19.6-kDa glycoprotein commonly used to treat
neutropenia (4, 5). Known sources of G-CSF in the body include
monocytes, mesothelial cells, fibroblasts, and endothelial cells,
and receptors for G-CSF are present on precursors and mature
neutrophilic granulocytes, monocytes, platelets, and endothelial
cells. At the myeloid progenitor cell level, G-CSF stimulates the
growth of neutrophil granulocyte precursors (6). G-CSF crucially
regulates survival of mature, i.e., postmitotic, neutrophils (7) by
inhibition of apoptosis (8).
We have recently uncovered the neuroprotective potential of
G-CSF in an acute stroke model (9). Here, we explore the mecha-
nisms responsible for that property and report on a dual func-
tionality of G-CSF in the brain that parallels its activity in the
hematopoietic system: inhibition of programmed cell death and
stimulation of neuronal progenitor differentiation.
G-CSF has robust neuroprotective activity in 2 different rodent stroke mod-
els and crosses the blood-brain barrier. We recently reported infarct-
reducing activity of G-CSF in the acute stroke model middle cere-
bral artery occlusion (MCAO) in rats when treatment was initiated
30 minutes after onset of ischemia (9). When we initiated treat-
Nonstandard?abbreviations?used: BBB, blood-brain barrier; bFGF, basic FGF;
CCA, common carotid artery; DCX, doublecortin; DIV, days in vitro; GFAP, glial
fibrillary acidic protein; MCA, middle cerebral artery; MCAO, MCA occlusion; NSE,
neuron-specific enolase; NSS, neurological severity score; PARP, poly-ADP ribose
polymerase; PLP, proteolipid protein; SVZ, subventricular zone; TTC, 2,3,5-triphenyl
Conflict?of?interest: A. Schneider, C. Krüeger, D. Weber, C. Pitzer, R. Laage, and
A. Bach are employees of Axaron Bioscience AG. R. Kollmar, S. Schwab, C. Sommer,
W.-R. Schäbitz, A. Schneider, C. Krüeger, D. Weber, M.H. Maurer, and N. Gassler are
inventors on a patent application incorporating parts of the presented findings.
Citation?for?this?article: J. Clin. Invest. 115:2083–2098 (2005).
2084? The?Journal?of?Clinical?Investigation http://www.jci.org Volume 115 Number 8 August 2005
ment with G-CSF (60 µg/kg body weight i.v.) in a delayed fashion,
2 hours after onset of ischemia, we still observed that the infarct
volume was robustly reduced, by 37% compared with that of
vehicle-treated rats (P < 0.01; Figure 1A). Mortality in the MCAO
model was 27% in the control group (4 of 15) and 15% in the
G-CSF–treated group (2 of 13). Moreover, in another model of cere-
bral ischemia, direct distal MCAO (10), G-CSF at a similar dose (50
µg/kg body weight i.v.) given 1 hour after occlusion onset resulted
in an infarct volume reduction of 42% (P < 0.05; Figure 1B). This
infarct reduction correlated with behavioral improvements in
the distal MCAO model (Figure 1C). In both models used, there
was no difference in the physiological parameters obtained, such
as blood pressure as well as blood glucose and blood gas levels,
and the extent of drop in cerebral perfusion as monitored by laser
Doppler flowmetry (data not shown). As expected, G-CSF treat-
ment at a dose of 60 µg/kg body weight in rats subjected to MCAO
led to about 2-fold elevations in leukocyte counts (neutrophils
and monocytes) after 24 hours (Supplemental Figure 1; supple-
mental material available online with this article; doi:10.1172/
JCI23559DS1). In summary, G-CSF had a stable neuroprotective
effect in 2 different infarct models.
A prerequisite for a direct action of G-CSF on the brain would
be penetration of the blood-brain barrier (BBB). We determined
the amount of iodinated G-CSF (131I–G-CSF) in brain and serum
at 1, 4, and 24 hours after i.v. injection in noninjured rats and cal-
culated the brain/serum ratios of 131I–G-CSF and 131I-albumin as
an index of BBB permeability. At every observation point, G-CSF
showed a higher brain/serum ratio, which indicated passage of
G-CSF through the intact BBB (Figure 1D).
G-CSF receptor and ligand are neuronally expressed and induced by
cerebral ischemia. The G-CSF receptor showed a broad, predomi-
nantly neuronal expression throughout the rat brain, with particu-
larly high immunopositivity in large principal neurons. Among
brain regions, there was a high expression in the cortex (most
pronounced in layers II and V) (Figure 2A), the hippocampus, the
subventricular zone (SVZ), the cerebellum (particularly in Purkinje
cells) (Figure 2B) as well as deep cerebellar (Figure 2C) and brain-
stem nuclei plus the mitral cells in the olfactory bulb. Importantly,
a corresponding neuronal staining pattern was also confirmed for
human (Figure 2D: frontal cortex, layer V) and mouse (data not
shown) brain tissue. Also, specificity of the G-CSF receptor signal
was demonstrated in neural and extraneural tissues by preincuba-
tion with the target peptide (Supplemental Figure 2). In a search for
sources for the ligand in the CNS, we found expression of G-CSF in
all brain regions where its receptor was expressed. For example, we
observed strong expression in the hippocampus CA3 field (Figure
2E), the hilus and subgranular zone of the dentate gyrus (Figure
2E, arrows), neurons in the entorhinal cortex (Figure 2F), neurons
in the olfactory bulb (Figure 2G), several cerebellar and brainstem
nuclei (Figure 2H), and cells in the SVZ (Figure 2I). Positive con-
trol stainings using the identical staining protocol and antibody
employed in Figure 2, F–I, yielded the expected published staining
patterns for G-CSF in extraneural tissues (Supplemental Figure 3).
In addition, neuronal expression was also detected using a second,
unrelated antibody against G-CSF (Supplemental Figure 4).
We then performed a series of experiments to confirm the true
neuronal expression of this secreted protein. As 1 report mentioned
described G-CSF expression in stimulated astrocytes in vitro (11),
we performed double-immunohistochemistry with the astro-
cytic marker glial fibrillary acidic protein (GFAP). There was no
appreciable astrocytic expression of G-CSF detectable in vivo in all
brain regions examined, both in noninjured and ischemic brains.
Figure 3 shows examples of the dentate gyrus hilus (Figure 3A)
and cortex (Figure 3B). In contrast, costaining with the neuronal
marker NeuN demonstrated perfect colocalization with G-CSF
(Figure 3C). Moreover, the pattern of G-CSF mRNA detection in
neurons of brain areas by in situ hybridization corresponded to
the staining pattern obtained by immunohistochemistry, which
confirmed neuronal synthesis of G-CSF (Figure 3, D–I). Note the
distinct staining in selected cells in the subgranular zone of the
dentate gyrus (Figure 3H). Finally, we performed laser-capture
microdissection of neurons and astrocytes from the mouse frontal
cortex and assayed these samples for G-CSF mRNA expression by
PCR. Indeed, using this highly sensitive approach, we could readily
detect G-CSF expression in the neuronal sample but not in astro-
cytes, even after 50 cycles of PCR amplification (Figure 3J). Thus,
G-CSF is a neuronally expressed protein in the CNS.
Astonishingly, G-CSF localized to neurons expressing its recep-
tor in all areas examined (Figure 4), which suggests an autocrine
activation mechanism of the receptor. We asked whether this
ligand/receptor system reacts to cerebral ischemia. Indeed, after
G-CSF has stable neuroprotective activity in focal cerebral ischemia
and passes the intact BBB. (A) G-CSF has efficacy in the transient
MCAO stroke model when given 2 hours after onset of ischemia, as
shown by reduction in infarct volume (dose: 60 µg/kg i.v.; vehicle, n = 7;
G-CSF, n = 10; **P < 0.01 by 2-sided t test). (B) G-CSF reduces infarct
volume in the rat cortical combined CCA/distal MCA occlusion model
when given 1 hour after onset of ischemia (dose: 50 µg/kg i.v.; n = 5
each; *P < 0.05). (C) Behavioral measurements in the cortical combined
CCA/distal MCA occlusion model. G-CSF–treated animals have a better
composite neurological deficit score (NDS) (*P < 0.05). (D) Comparison
of the brain/serum ratios of i.v. injected iodinated G-CSF and albumin at
1, 4, and 24 hours following injection. Albumin does not pass the BBB.
Radiolabeled proteins (G-CSF and BSA) were injected via the tail vein
of healthy female Sprague-Dawley rats. The relative amount of radiola-
beled G-CSF and BSA in serum and brain was measured, and the ratio
of brain/serum was plotted against the time. The brain/serum ratio of
G-CSF was significantly greater than that of albumin, which indicated
passage of the intact BBB in non-ischemic animals.
? The?Journal?of?Clinical?Investigation http://www.jci.org Volume 115 Number 8 August 2005
2 hours reperfusion in the MCAO stroke model, there was a dra-
matic upregulation of the ligand on the ipsilateral forebrain hemi-
sphere (more than 100-fold according to quantitative PCR; Figure
5A) that was accompanied by induction of G-CSF mRNA on the
contralateral hemisphere. At 6 hours following ischemia, this induc-
tion became more specific to the ischemic hemisphere, and it was
no longer detectable at 20 hours of reperfusion. This upregulation
of the ligand was accompanied by a more modest induction of the
G-CSF receptor at 6 hours (Figure 5B), more prominent in the
ipsilateral than the contralateral hemisphere. A more distinct induc-
tion of G-CSF receptor mRNA at 6 hours was seen in periinfarct cor-
tex samples from a rat cortical photothrombotic model (Figure 5C).
In addition, we could also detect ligand induction in a global model
of cerebral ischemia (Supplemental Figure 5).
This mRNA upregulation by cerebral ischemia was confirmed on
the protein level by immunohistochemistry at 6 hours following
ischemia (Figure 5, D–O). Induction of receptor and ligand was
most clearly seen in the periinfarct area, e.g., in the MCAO model
(Figure 5, D [receptor] and G [ligand]), or in cortical photothrom-
botic ischemia, where the infarct borders are easily recognizable
(Figure 5, J [receptor] and M [ligand]). In conclusion, G-CSF and
its receptor are coexpressed in neurons in the rodent CNS and are
upregulated by ischemic stimuli.
We then asked whether these data from the rodent are likely rel-
evant to the human system. Indeed, when comparing the cortical
periinfarct area from a human stroke case 3 days after stroke onset
(Figure 6A) with the corresponding contralateral cortex (Figure
6B) or a neuropathologically normal matched control brain (Fig-
ure 6C), we found clear induction of G-CSF receptor expression
in neurons (see insets), which suggests comparable activity of the
G-CSF system in the human brain.
G-CSF activates antiapoptotic pathways in cultured neurons. As
G-CSF blocks apoptosis in cells of the myeloid lineage, we
hypothesized that G-CSF might also interfere with programmed
cell death in neurons. Cortical neurons from rat cortex at E18
invariably expressed the G-CSF receptor (Figure 7A). G-CSF pro-
tected cortical neurons against programmed cell death caused
by the apoptosis inducer camptothecin (Figure 7B). This activity
appeared to be mediated via the neuronal G-CSF receptor, as an
antibody against the receptor was able to abolish protection (Fig-
ure 7C). Activity of G-CSF against neuronal cell death resulting
from other apoptosis-inducing agents could also be observed.
An apoptotic stimulus for neurons with high relevance to stroke
pathophysiology is NO. G-CSF reduced NO-induced poly-ADP
ribose polymerase (PARP) and caspase-3 cleavage in primary neu-
rons (Figure 7D). This antiapoptotic activity was not specific to
cells of rodent origin but could also be seen in NO-challenged
human SHSY-5Y neuroblastoma cells (Figure 7E), which also
expressed the G-CSF receptor (data not shown). We therefore
analyzed the activation of antiapoptotic pathways after G-CSF
stimulation in primary cortical neurons.
One important known transduction factor of G-CSF in the
hematopoietic system is STAT3. Although activation of STAT3
by phosphorylation was detected in neurons after 5 minutes of
G-CSF exposure (Figure 8, A and B), this induction was rather
moderate and very transient. STAT3 is phosphorylated by the
JAK2 kinase, which is recruited to the intracellular domains of the
G-CSF receptor upon ligand binding. STAT3 activation appeared
to be specifically mediated via the known pathway involving the
G-CSF receptor present on neurons (Figure 8A, bottom lane), as
AG490, a specific JAK2 inhibitor, strongly reduced STAT3 phos-
phorylation 5 minutes after addition of G-CSF (Figure 8A, right).
Typical for the kinetics of G-CSF–activated STAT3 in the hemato-
poietic lineage (12), STAT phosphorylation decreased rapidly over
a time course of 60 minutes (Figure 8B). G-CSF also led to a long-
lasting (at least 8 hours), but overall moderate increase in protein
The G-CSF receptor (A–D) and its ligand (E–I) are expressed by
neurons in a variety of brain regions in the rat. Among other areas,
expression of the receptor was detected in pyramidal cells in corti-
cal layer V (A); Purkinje cells in the cerebellum (B); and cerebellar
nuclei (C). Importantly, this neuronal staining pattern could also be
detected in the human brain (frontal cortex, D). G-CSF is expressed by
neurons in many areas of the CNS. Immunohistochemistry identifies
G-CSF–positive cells in the CA3 region of the hippocampus (E) and
the subgranular zone and hilus of the dentate gyrus (E, arrows), the
entorhinal cortex (F), the olfactory bulb (G), and cerebellar nuclei (H).
Expression was also seen in cells in the SVZ (I).
2086? The?Journal?of?Clinical?Investigation http://www.jci.org Volume 115 Number 8 August 2005
levels of the STAT3 target Bcl-XL, a potent antiapoptotic factor in
neurons (13) (Figure 8C).
Next, we determined activation levels of the ERK family of kinas-
es. ERK1/2, which has been linked to both pro- and antiapoptotic
events in neurons (e.g., refs. 14, 15) was only weakly and transiently
activated by G-CSF (Figure 8D). In contrast, the newly described
ERK5 kinase demonstrated a strong and lasting activation pattern
(Figure 8D, bottom 2 rows). Interestingly, a recent report connects
ERK5 activation to survival signals elicited by trk receptors (16).
One of the most potent antiapoptotic transduction pathways
in all cell types including neurons known to date is the PI3K/Akt
pathway (17). Akt is activated via PI3K and 3′-phosphoinosit-
ide–dependent protein kinase (PDK), and the amount of active
Akt can be determined based on Ser437 phosphorylation. In
untreated neurons, there was only a faint band visible cor-
responding to phosphorylated Akt (Figure 8E, bottom 2
rows). However, 5 minutes after G-CSF exposure, levels
of phosphorylated Akt dramatically increased, and they
remained elevated for at least 1 hour. The kinetics of Akt
activation following G-CSF exposure corresponded well
with the phosphorylation of PDK1, the protein kinase
immediately upstream of Akt in the PI3K/Akt pathway
(Figure 8E, top 2 rows). The phosphorylation of Akt 5
minutes after addition of G-CSF was completely blocked
by the PI3K inhibitor LY294002 (Figure 8E, right). Thus,
Akt is a prominent signal induced by G-CSF in neurons
and appears to be activated via the known PI3K/PDK path-
way originating at the G-CSF receptor. Inhibition of PI3K by
LY294002 was able to partially block G-CSF–mediated protection
against apoptosis in neurons (Figure 8F) or in human neuroblas-
toma cells (Figure 8G), which suggests that Akt activation indeed
is a crucial factor in G-CSF’s antiapoptotic activity.
In conclusion, these results indicate that G-CSF counteracts pro-
grammed cell death in neuronal cells, an activity that is at least
partially mediated by the PI3K/Akt pathway.
G-CSF drives neuronal differentiation in vitro. We noted that G-CSF
receptor and ligand were expressed in the dentate gyrus by neu-
rons of the subgranular zone and the hilus region (for examples,
see Figure 2E, arrows and Figure 3, H and I). Expression was also
noted in cells of the SVZ (Figure 2I). As these regions are known
G-CSF is specifically expressed by neurons in the rat CNS.
(A–C) Double-immunofluorescence staining with the astrocytic
marker GFAP revealed absence of G-CSF expression in astro-
cytes in the hilus of the dentate gyrus (A) or cortex (Cx; B). In
contrast, there was perfect colocalization of G-CSF with cells
expressing the neuronal marker NeuN (C). (D–I) In situ hybrid-
ization confirmed the neuronal expression of G-CSF and dem-
onstrated an expression pattern that paralleled results obtained
by immunohistochemistry. For example, G-CSF mRNA was
detected in pyramidal neurons in the cortex (D; original magnifi-
cation, ×40), in the hippocampus CA3 field (F; original magnifi-
cation, ×40), and in specific cells located in or near the subgran-
ular zone in the dentate gyrus (DG) (H; original magnification,
×40). Sense probes did not yield any specific staining in corre-
sponding sections (E, G, and I; original magnification, ×40). (J)
Using amplified mRNA from laser-excised neurons or astrocytes
from the mouse cortex (100 cells each), a G-CSF–specific PCR
signal could only be obtained in the neuronal pool but not from
astrocytes after 50 amplification cycles. As a control, GFAP
was amplified only from the astrocytic population, whereas the
ubiquitous housekeeping gene cyclophilin was amplified from
both cell pools. A brain cDNA library served as positive control
(Pos.) for all PCR reactions. PCR reactions using water as input
served as negative control (Neg.). M, size marker.
The G-CSF receptor and its ligand colocalize in neurons in
the cortex. Double-immunofluorescence detected G-CSF
receptor (G-CSFR) (A) and G-CSF itself (B) in identical layer
V neurons in the frontal cortex. Note the relatively stronger
presence of the receptor in dendritic processes (C).
? The?Journal?of?Clinical?Investigation http://www.jci.org Volume 115 Number 8 August 2005
to harbor neuronal progenitor cells and G-CSF has a potent role
in progenitor cell differentiation in the hematopoietic system, we
asked whether G-CSF might have a functional role in differentia-
tion of adult neural stem cells. Indeed, adult neural stem cells iso-
lated from the rat SVZ or hippocampal region that grow as neuro-
spheres in culture expressed the G-CSF receptor at the mRNA level
(Figure 9A). By immunocytochemistry we could also detect colocal-
ization with the stem cell marker nestin (Figure 9B). We therefore
examined the effects of G-CSF treatment on adult neural stem cells.
G-CSF dose-dependently induced activity of the promoter of the
mature neuronal marker β-III-tubulin (Figure 9C) with a maximal
induction greater than that reached by the most standard neuronal
induction protocol (addition of FCS and withdrawal of EGF and
basic FGF [bFGF]) (Figure 9C, far right bar). We corroborated this
result by measuring expression levels of cell type–specific differ-
entiation markers (nestin, β-III-tubulin, neuron-specific enolase
[NSE], proteolipid protein [PLP], and GFAP) by quantitative
PCR after 4 days of G-CSF treatment. G-CSF again led to a dose-
dependent increase in the expression of markers for neuronal
differentiation (β-III-tubulin and NSE) and a slight increase in
the expression of mature glial markers (PLP, GFAP; Figure 9D).
The expression of nestin stayed constant with increasing con-
centrations of G-CSF, which indicated that G-CSF treatment did
not diminish the pool of undifferentiated cells (Figure 9D). As a
third approach to assay neuronal differentiation, we used FACS
analysis to determine the number of microtubule-associated
protein 2–positive (MAP2-positive) cells after G-CSF treatment.
Also in this assay, G-CSF led to an increase in the population of
cells expressing mature neuronal markers (Figure 9E). Thus, the
G-CSF system has a functional role in the regulation of differen-
tiation of adult neural stem cells in vitro.
G-CSF improves functional outcome after cerebral ischemia. To
determine whether these basic properties of G-CSF had con-
sequences for long-term postischemic behavioral changes and
for neurogenesis in vivo, we designed an experiment wherein
G-CSF was given for 5 consecutive days at a dose of 15 µg/kg body
weight following photothrombotic induction of ischemia in the
sensorimotor cortex. This model has the advantage of producing
defined neurological deficits without affecting survival. The dos-
age chosen is similar to the clinically used regimen in neutropenic
patients (10 µg/kg/d for up to 14 days).
Sensorimotor deficits were obvious in vehicle-treated ischemic
animals compared with sham-operated rats in the rotarod and
adhesive tape removal test, and in the neurological severity score
(NSS), which included the results of the beam balance test, for
up to 6 weeks after the insult (Figure 10, A–D, compare red [isch-
emia + vehicle] and green [sham]). G-CSF–treated ischemic rats
performed significantly better in all test paradigms than vehicle-
treated animals when group means per time point were compared
G-CSF and its receptor are induced by cerebral ischemia. (A–C)
Quantitative PCR demonstrates induction of mRNA following cere-
bral ischemia. (A) In the MCAO model, G-CSF mRNA is induced
more than 100-fold in the ipsilateral and contralateral forebrain hemi-
sphere at 2 hours following ischemia. At 6 hours, induction levels
dropped, and overexpression became more specific to the ipsilateral
hemisphere. At 20 hours, induction was no longer detectable (data
not shown). (B) Moderate induction of the G-CSF receptor mRNA
in forebrain hemispheres was seen 6 hours following MCAO. (C)
Receptor induction was also detected 6 hours after ischemia in
another ischemic model, cortical photothrombotic ischemia in biopsy
material from the periinfarct cortex. The substantially higher induc-
tion reflects the strong induction in the infarct penumbral zone.
(D–O) Immunohistochemical detection of receptor and ligand in the
corresponding ischemia models. (D–I) Staining for G-CSF recep-
tor (D–F) and ligand (G–I) in the MCAO model, 6 hours after isch-
emia: ipsilateral cortex (D and G) and corresponding areas of the
contralateral hemisphere (E and H) and the cortex of a sham-operat-
ed rat (F and I). (J–O) Staining for G-CSF receptor (J–L) and G-CSF
itself (M–O) in the photothrombotic model: ipsilateral cortex (J and
M) and corresponding areas of the contralateral hemisphere (K and
N) and the cortex of a sham-operated rat (L and O). The infarct bor-
der zone is shown in the upper-right quadrant in D, G, J, and M and
is particularly clear in the photothrombotic model. Note the strong
dendritic staining for the G-CSFR. Original magnification ×20.
2088? The?Journal?of?Clinical?Investigation http://www.jci.org Volume 115 Number 8 August 2005
(Figure 10, A–D, red and blue curves). We also used a statistically
more appropriate test for time-series measurements in subjects
and compared areas under the individual curves over time (18)
(Figure 10, A–D, bar graphs). This analysis also demonstrated a
robust behavioral improvement upon G-CSF compared with vehi-
cle treatment (compare red and blue bars). As expected, the perfor-
mance deficit induced by ischemia in the adhesive tape removal
paradigm was more pronounced on the side contralateral to the
lesion (Figure 10, C and D; compare red curves). G-CSF induced
an improvement of performance both on the contralateral, paret-
ic forepaw and on the ipsilateral extremity (Figure 10, C and D;
compare blue versus red curves). However, likely owing to the
less pronounced deficit, this difference was only significant for
2 time points on the ipsilateral side but not in the AUC analysis
(P = 0.13; Figure 10D). No difference was noted between sham-
operated G-CSF– and vehicle-treated animals (data not shown).
Physiological parameters (rectal temperature, pH, partial pressure
of CO2 [pCO2], pO2, and mean arterial pressure during surgery)
did not differ between vehicle- and G-CSF–treated ischemic rats.
Also, there were no differences in mortality or body weight among
all groups (data not shown). Therefore, G-CSF treatment leads to
long-term behavioral improvements after cerebral ischemia.
G-CSF stimulates neural progenitor cells in vivo. We analyzed progeni-
tor cells of the lateral ventricle wall by immunofluorescence against
doublecortin (DCX), a microtubule-associated protein that is spe-
cifically expressed in neural progenitor cells and immature neu-
rons (19, 20). The development into new neurons was detected by
colabeling of DCX with the mature neuronal
marker NeuN (21).
Migration of neuronal progenitor cells
from the lateral ventricle wall to the lesioned
neocortex has previously been reported (22,
23). When we analyzed the distribution of
DCX-expressing cells in the ventricle wall as
well as overlying corpus callosum and cortex,
we found a visible recruitment of progenitor
cells into the ischemic area of the neocor-
tex. This response was visibly enhanced by
peripheral infusion of G-CSF (Figure 11,
A–I). Area and intensity of DCX immunoreac-
tivity were significantly increased in ischemic
G-CSF–treated (ischemia + G-CSF) animals
compared with vehicle-treated (ischemia +
vehicle) animals (area by 300% and intensity by 225%; P < 0.05). As
an indicator of ongoing neuronal differentiation of the DCX-posi-
tive cells, coexpression with NeuN was frequently detected in cells
surrounding the lesion site (see Supplemental Figure 6 for Z-stack
analysis). However, using BrdU injections during the first 5 days
after ischemia to label newly generated cells, we found that BrdU
was not incorporated into NeuN-expressing cells in the cortical
areas surrounding the lesion site.
The striatum has previously been described to have some degree
of progenitor activation and neurogenesis after ischemia due to its
proximity to the subventricular pool of neural stem and progeni-
G-CSF receptor is neuronally induced upon human stroke. Immunohistochemical detec-
tion of G-CSF receptor in the ipsilateral (A) and contralateral (B) cortex of a human brain
obtained at autopsy 3 days after onset of ischemic stroke as well as the frontal cortex of
an age-matched neuropathologically normal control brain (C). Scale bars: 100 µm. Note
increased staining of neurons in the ipsilateral cortex compared with the contralateral cortex
and control brain (insets).
G-CSF counteracts programmed cell death in rat cortical neurons
(A–D) and human neuroblastoma cells (SHSY-5Y) (E) in vitro. (A) The
G-CSF receptor is present on primary cortical neurons in culture as
shown by immunocytochemistry. (B) G-CSF of both human (h) and
mouse (m) origin counteracts camptothecin-induced programmed cell
death in primary neurons as determined by caspase-3/7 activity. (C)
Preincubation of primary neurons with an antibody against the G-CSF
receptor abolishes the antiapoptotic activity of G-CSF (not signifi-
cant). (D) The NO donor NOR3 [(±)E)-4-ethyl-2-[(E)-hydroxyimino]-5-
nitro-3-hexenamide] (150 µM) induces apoptosis in primary neurons
as evidenced by PARP and caspase-3 cleavage (immunoblots, first
and second lanes), which is reduced by G-CSF treatment (third lane).
(E) Also in the human neuroblastoma cell line SHSY-5Y, human or
mouse G-CSF reduces caspase activation by the NO donor NOR3.
Bar graphs show relative caspase activity levels after normalization to
control values. Rel. units, relative units.
? The?Journal?of?Clinical?Investigation http://www.jci.org Volume 115 Number 8 August 2005
tor cells. We analyzed DCX-positive cells in the striatum at 6 weeks
after cortical photothrombosis and peripheral G-CSF infusion.
Although the data suggest a trend toward more DCX-positive cells
in the striatum of ischemia + G-CSF animals, no significant differ-
ences between ischemia + vehicle and ischemia + G-CSF animals
were detected (data not shown).
The hippocampus has substantial renewal capacity for
granule cells throughout the life of the rat. Since several
studies have shown that hippocampal neurogenesis is
upregulated after global and focal ischemia (2, 24–27),
we investigated whether G-CSF treatment would alter
the response of dentate gyrus progenitor cells to cortical
injury. Using BrdU injections on days 1–5 after cortical
photothrombosis, we determined the number of cells
positive for BrdU and NeuN (Figure 12, A–E).
When looking at the total number of BrdU+ cells in the
hippocampus, we noticed an expected strong increase
in vehicle-treated animals subjected to cortical photo-
thrombosis on the side of the infarct (ipsilateral + vehi-
cle) but also contralaterally (contralateral + vehicle) (Fig-
ure 12F, compare sham-operated, vehicle-treated [sham
+ vehicle] with ipsilateral + vehicle and contralateral +
vehicle). Although there was a slight increase in BrdU+
cells in the ipsilateral dentate gyrus upon G-CSF treat-
ment (Figure 12F, ipsilateral + vehicle and ipsilateral +
G-CSF), this was not statistically significant. However,
G-CSF induced a significant rise in newly generated cells
in the dentate gyrus in sham-operated animals (Figure
12F, sham + vehicle vs. sham + G-CSF).
Counting NeuN/BrdU-double-positive cells, we
found that G-CSF indeed increased the number of
newly generated granule cells after ischemia on the
side of the lesion (Figure 12G, ipsilateral + vehicle vs.
ipsilateral + G-CSF; P < 0.01). In the contralateral (unle-
sioned) dentate gyrus, the increase in newly generated
granule cells after G-CSF treatment was smaller and was
not statistically significant (Figure 12G, contralateral
+ vehicle vs. contralateral + G-CSF). However, G-CSF
significantly increased neurogenesis in sham-operated,
nonischemic animals (Figure 12G, sham + vehicle vs.
sham + G-CSF; P < 0.05). Thus, peripheral administra-
tion of G-CSF increases hippocampal neurogenesis
not only in ischemic animals, but also in the intact,
Here we have demonstrated that the hematopoietic
factor G-CSF is an endogenous, neuronally expressed
ligand that is upregulated upon ischemia and provides
protection against programmed cell death in neurons,
which is reflected by robust neuroprotective activity
in acute stroke models in vivo. In addition, G-CSF
displays a strong neurogenic potential in vitro and in
vivo, corresponding to long-term behavioral improve-
ments after ischemia.
We found that G-CSF is expressed by neurons in
many areas of the CNS, which implies important new
functions of this protein in the CNS. This discovery is
particularly surprising as G-CSF is a long-known pro-
tein that was cloned many years ago as a growth fac-
tor in the hematopoietic system (28). To our knowledge, the only
reports that suggest expression of G-CSF in neural cell types deal
with stimulus-induced expression in astrocyte cultures (11, 29).
We have therefore carefully examined the cellular origin of G-CSF
and could not detect any astrocytic G-CSF expression in vivo, even
in the acute cerebral ischemia paradigms studied. It is, however,
Signal transduction events evoked by G-CSF treatment of rat cortical neurons.
G-CSF activates STAT3, ERK, and PI3K/Akt pathways in primary cortical neu-
rons. Western blots for phosphorylated proteins were stripped and reprobed with
antibodies nonselective for phosphorylation. (A) STAT3 was rapidly but moder-
ately phosphorylated (pSTAT3) 5 minutes after addition of G-CSF to the medium.
Neuronal expression of the G-CSF receptor was also confirmed by Western blot
analysis. Addition of the JAK2 inhibitor AG490 inhibited hyperphosphorylation of
STAT3 5 minutes after addition of G-CSF. (B) Quantification of phosphorylation
ratios from Western blots illustrates the moderate and transient but reproducible
activation of STAT3 by G-CSF (data from 3 independent experiments). (C) G-CSF
leads to increase of protein levels of the antiapoptotic STAT3 target Bcl-XL in pri-
mary cortical neurons over 8 hours. (D) Induction of ERK1/2 (double band) and
ERK5 by G-CSF. While ERK1/2 activation appears to be very transient (upper
rows), ERK5 is induced for at least 60 minutes following G-CSF exposure (lower
rows). (E) Stable induction of Akt phosphorylation upon addition of G-CSF to the
medium (50 ng/ml) shown by immunoblotting with a Ser437 phosphorylation–spe-
cific antibody. In accordance with the known Akt activation pathway, the PDK kinase
upstream of Akt was phosphorylated, and Akt phosphorylation could be blocked by
preincubation of the neurons with the PI3K inhibitor LY294002. (F and G) Inhibition
of PI3K by LY294002 diminishes G-CSF–mediated protection from apoptosis (mea-
sured by luminometric caspase-3/7 activity assay) in rat cortical neurons treated
with staurosporine (F) or in the human neuroblastoma cell line SHSY-5Y, where cell
death was elicited by camptothecin (G). Bars indicate mean relative protection (%)
against the cell death stimulus ± SEM; values were normalized to the appropriate
controls (n = 16 each).
2090? The?Journal?of?Clinical?Investigation http://www.jci.org Volume 115 Number 8 August 2005
certainly possible that G-CSF expression in astrocytes might be
evoked in vivo by other stimuli or occur under different ischemic
conditions or at different postischemic time points.
Neuronally expressed G-CSF was induced more than 100-fold at the
level of transcription by focal cerebral ischemia. To the best of our
knowledge, this is the strongest regulation of any gene by ischemic
events in the brain that has been reported so far, implicating an impor-
tant adaptive response in neurons. In the MCAO model, we found
transcriptional induction of this protein early after ischemia in both
hemispheres, a phenomenon frequently encountered in this model
(30). However, induction became more specific to the ipsilateral hemi-
sphere at 6 hours and was transient, with elevated mRNA levels no
longer detectable at 20 hours following ischemia. Importantly, induc-
tion was seen not only in focal but also in global ischemic models.
G-CSF’s actions in the brain appear to be specifically medi-
ated through the G-CSF receptor, which has an astonishingly
broad, predominantly neuronal expression pattern
in the CNS. Indeed, G-CSF localized to neurons
expressing its receptor. Moreover, the G-CSF recep-
tor itself was induced at 6 hours after ischemia.
Immunohistochemistry demonstrated a strong induc-
tion in the periinfarct zone of both receptor and ligand
at this time point. The periinfarct zone is known to
harbor neurons at risk of dying, which suggests that
G-CSF and its receptor likely function as an autocrine
adaptive system in neurons. An autocrine signaling
mechanism has indeed been discussed for a number
of neuroprotective growth factors in the brain, such as
brain-derived neurotrophic factor (BDNF; 31), eryth-
ropoietin (EPO; 32), VEGF (33), and neurotrophin-3 (NT-3; 34).
Systemically given G-CSF was able to pass the intact BBB, a
property that is shared with other hematopoietic factors such as
EPO (35) and GM-CSF (36), and was neuroprotective in 2 different
models of focal cerebral ischemia. In vitro, G-CSF displayed strong
antiapoptotic activity in neuronal cells. G-CSF evoked very mod-
est and transient activation of STAT3 and ERK1/2 and a strong
lasting activation of ERK5, which has recently been implicated in
promoting neuronal survival (16). ERK5 was also shown to be acti-
vated by G-CSF in non-neural cell types (37). However, the most
dramatic effect of G-CSF was seen on the PI3K/Akt pathway, and
inhibition of PI3K indeed interfered with the antiapoptotic activ-
ity of G-CSF. Since the original description of antiapoptotic activi-
ties of Akt in neurons (17), a number of reports have confirmed
the powerful central regulatory role of this kinase for neuronal
survival (38). In hematopoietic cells, G-CSF activates intracellular
The G-CSF receptor is present on adult neural stem cells
(NSCs) and drives neural progenitor differentiation in
vitro. (A) Results of PCR analysis for the G-CSF recep-
tor on neural stem cells demonstrate presence of G-CSF
receptor mRNA in neurospheres in culture. (B) Double-
fluorescence immunocytochemistry on neural stem cells
plated onto coated 96-well plates. Almost all cells were
positive for the stem cell marker nestin and for the G-CSF
receptor (original magnification, ×20). (C–E) G-CSF drives
neuronal phenotype induction in adult neural stem cells
in vitro. (C) As 1 approach, we assayed β-III-tubulin pro-
moter activity by a luciferase reporter assay. Treatment
with G-CSF in increasing concentrations for 48 hours
increased promoter activity. As a positive control, neu-
ral stem cells were treated with the standard differentia-
tion protocol involving withdrawal of EGF and bFGF and
addition of FCS (+FCS/–EGF/–bFGF). (D) G-CSF treat-
ment induces a concentration-dependent upregulation of
the neuronal markers NSE and β-III-tubulin 4 days after
G-CSF treatment, as indicated by quantitative PCR. The
glial markers PLP and GFAP are moderately induced in
response to increasing G-CSF concentrations. Error bars
indicate standard deviations calculated from measure-
ments done with serial dilutions of the cDNA samples
(1:3, 1:9, 1:27, and 1:81). (E) FACS analysis for MAP2-
positive cells served to confirm the data presented above
on an individual cell basis. Cells were treated for 4 days
prior to analysis. Left: Examples of FACS runs of vehicle-
treated and G-CSF–treated (100 ng/ml) neural stem cells.
Right: The bar graph summarizes the results of several
experiments (vehicle, n = 4; G-CSF, n = 7; P < 0.005).
?The?Journal?of?Clinical?Investigation http://www.jci.org Volume 115 Number 8 August 2005
signaling pathways including STAT3 (39) and Akt (40), which are
both linked to suppression of apoptosis and proliferation. There-
fore, G-CSF signaling and its role in suppressing apoptosis seem
to be preserved both in cells of the hematopoietic lineage and in
neurons. The involvement of the antiapoptotic PI3K/Akt pathway
in neurons is most likely 1 crucial mechanism for the robust acute
infarct volume–reducing effect of G-CSF.
In the hematopoietic system, G-CSF’s functions are dual, as they
involve inhibition of apoptosis but also differentiation of hema-
topoietic stem cells. This function seems to be preserved in the
CNS, as we found expression of receptor and ligand on adult neu-
ral stem cells and induction of a neuronal phenotype in these cells
by addition of G-CSF in vitro. G-CSF induced functional recovery,
which correlated with increased neuronal progenitor activation in
the periphery of the ischemic lesion (cortex and corpus callosum)
and with enhanced neurogenesis in the dentate gyrus.
Cortical lesions have previously been reported to enhance
neuronal progenitor proliferation in the lateral ventricle wall.
Similarly, we have observed that the amount of DCX-positive cells
outside the ventricle wall and rostral migratory stream are visibly
enhanced in lesioned animals as compared with sham-lesioned
controls. We observed an additional increase in the amount of
DCX-positive cells under G-CSF treatment that were in close prox-
imity to the lesion site. Although a large number of DCX-positive
cells was detected in the immediate periphery of the lesion, the
colabeling of BrdU with mature neuronal marker did not indicate
substantial maturation of progenitor cells into neurons. Neuro-
genesis in the lesioned cortex has been reported only by Magavi
and colleagues (41), who used a very
selective apoptotic elimination of
individual cortical neurons, but not
by Arvidsson and colleagues (42), who
used an MCAO model. Neurogenesis
in the striatum was slightly increased
after G-CSF treatment, but again the
number of newly generated cells was
rather small. This corresponds to the
findings of Arvidsson et al. that dem-
onstrated that less than 0.2% of the
damaged striatal neuronal popula-
tion was replaced by newly generated
cells after MCAO (42).
The most striking effect of periph-
erally administered G-CSF on the
brain was seen in the dentate gyrus,
where G-CSF increased the number of
newly generated neurons under isch-
emic conditions but also in nonisch-
emic, sham-operated animals. It is
therefore intriguing to speculate that
G-CSF may enhance structural repair
and function even in healthy subjects
or at long intervals after stroke.
Generation of new differentiated
cells from stem cells involves an
intricate interplay among prolifera-
tion, differentiation, and selective
survival. In our in vitro experiments,
G-CSF induced neuronal differen-
tiation of adult stem cells without
apparently elevating the number of immature stem cells, at least
at the time points examined (see Figure 9D). In vivo, the signifi-
cant increase in hippocampal neurogenesis (BrdU+/NeuN+ cells,
Figure 12F) was based on the fact that a much higher fraction of
BrdU+ cells turned into granule cells in ischemic, G-CSF–treated
animals compared with ischemic, untreated animals, although the
total number of BrdU+ cells was unaltered (Figure 12G). This indi-
cates a predominant role of G-CSF in survival and differentiation
of progenitor cells in the postischemic brain.
Although more work needs to be done to better understand the
balance of effects evoked by G-CSF during neurogenesis in the adult,
it is likely that one basic property of G-CSF in neurons, antiapoptosis,
also plays a part in the observed neurogenesis. In the hematopoietic
system, counteraction of the built-in apoptosis program in progeni-
tor cells by G-CSF is intricately linked to self-renewal and generation
of mature cell types of the blood (43). In the neurogenic regions of
the adult brain, the majority of proliferative cells are eliminated by
apoptosis before reaching a mature phenotype (44–46). Moreover,
the involvement of apoptotic signaling in adult hippocampal neuro-
genesis has been highlighted by a recent study on Bax-deficient mice
(47). A combined mechanism of proliferative and survival-promot-
ing effects on adult brain progenitor cells in vivo has indeed been
proposed for the action of several other brain- and blood-derived
growth factors, such as BDNF and VEGF (48–51).
The cortical photothrombosis model used in this study has the
most prominent impact on sensorimotor behavior, which was also
measured in the test battery performed, whereas hippocampal for-
mation is most frequently linked to learning and memory processes.
G-CSF treatment improves long-term functional outcome after cortical ischemia. (A–D) G-CSF
significantly improved motor recovery as measured by rotarod performance (A) and NSS, which
included the results of the beam balance test (B), compared with those in nontreated, ischemic
control animals. Sensory-motor function as measured by adhesive tape removal was significantly
better in G-CSF–treated, ischemic animals compared with ischemic controls when the contralateral
forepaw was tested (C) and borderline significant in the ipsilateral forepaw (D). Bar graphs repre-
sent an analysis of area under the curve (AUC) for each rat over time in an experimental group.
*P < 0.05; **P < 0.01; #P < 0.001.
2092?The?Journal?of?Clinical?Investigation http://www.jci.org Volume 115 Number 8 August 2005
However, there is also a wealth of data supporting a possible role of
the hippocampus in functional recovery from motor deficits. The
hippocampus produces a slow-wave activity known as rhythmical
synchronous activity (τ rhythm), which is thought to be involved in
sensorimotor integration and movement initiation (52–54). Type
1 τ activity gives a direct indication of the level of activation of the
motor systems involved in voluntary motor activity, whereas type
2 τ activity indicates the processing of sensory information (52). In
the context of our experiments, sensorimotor integration is most
crucial to the adhesive-removal paradigm, where the treatment
effect of G-CSF was most prominent (see Figure 10C). Cortical
lesions have in fact been shown to disrupt the hippocampal τ activ-
ity patterns (55). The connection between the motor system and
hippocampal neurogenesis is further supported by the finding that
voluntary running is a strong activator of the latter in mice (56–58).
We therefore hypothesize that the observed G-CSF–induced
increase in hippocampal neurogenesis directly impacts recovery
from cortical lesion–induced sensorimotor deficits.
G-CSF signaling appears to be a novel protective system in the
brain that is involved in counteracting acute neurodegeneration and
regulating the formation of new neurons. G-CSF’s principal cellular
functions in the CNS appear to be remarkably similar its functions
in the hematopoietic system. Thus, G-CSF’s basic functions have
apparently been conserved and utilized in 2 different body compart-
ments. While the direct actions of G-CSF uncovered here appear suf-
ficient to fully explain the observed in vivo effects of this protein, it
is certainly possible that additional mechanisms such as the mobi-
lization of bone marrow stem cells have a role in G-CSF–mediated
neuroprotection, although proof for this hypothesis is lacking at
present (59, 60). We have concentrated here on examining the effects
of G-CSF in vivo by using the most clinically relevant application
scheme of peripheral administration. For the further dissection of
mechanisms of action, and to study the role of brain-endogenous
G-CSF, studies with transgenically modified mice, such as neuron-
specific knock-outs for the G-CSF receptor, are warranted.
In therapeutic terms, G-CSF fulfills the criteria of a novel type
of stroke drug discussed in the introduction. Its multimodality,
together with the ability to penetrate the BBB and its documented
history as a well-tolerated drug, make G-CSF an ideal drug can-
didate for treatment of stroke. Our data suggest a comparable
functionality of the neural G-CSF system in the human. First, the
G-CSF receptor is neuronally expressed in the human brain in a
pattern of distribution similar to that in rodents. Second, a human
neuroblastoma line expresses the receptor and is protected against
programmed cell death. Finally, the G-CSF receptor is induced in
the ipsilateral infarcted cortex shortly after stroke. We have there-
fore started a phase IIa trial to establish the safety of i.v. adminis-
tered G-CSF in acute stroke patients.
The broad expression of the G-CSF receptor in many brain areas,
the passage of G-CSF through the intact BBB, the effect on neuro-
genesis in the nonischemic animal, and the favorable tolerance pro-
file suggest that G-CSF might be beneficial for a number of other
neurodegenerative and psychiatric disorders in which neuronal
cell death and/or disturbances in neurogenesis are involved.
Intraluminal occlusion model. Animals received inhalation anesthesia with
70% N2O, 30% O2, and 1% halothane. The femoral artery was cannulated
G-CSF induces neural progenitor cells and their migration in subcortical areas. (A–I) G-CSF induced substantially more neural progenitor cells
and immature neurons (DCX in red) in subcortical regions adjacent to the ischemic lesion (NeuN in green, BrdU in blue). (A) Control: unlesioned
hemisphere of G-CSF–treated ischemic animals. (B) Lesioned hemisphere with G–CSF treatment. (C) Lesioned hemisphere with sham treatment.
Note in B that G-CSF induced a stream of DCX-positive cells migrating toward the ischemic lesion (upper right). (D–I) Details from the boxed areas
in A–C. Note the density of DCX expression in F. Images represent cumulative confocal image Z-stacks throughout the whole slice thickness.
?The?Journal?of?Clinical?Investigation http://www.jci.org Volume 115 Number 8 August 2005
for recording of continuous arterial blood pressure and blood sampling
for gas analyses. The right femoral vein was used for drug delivery. During
the experiment, core body temperature was monitored and maintained at
37°C by a thermostatically controlled heating pad (FMI GmbH). MCAO
was induced with a silicon-coated (Provil Novo; Heraeus Kulzer) 4-0 nylon
filament (ETHICON) that was introduced into the common carotid artery
(CCA) and advanced into the internal carotid artery as described previously
(9). Successful MCAO was verified by laser Doppler flowmetry (Perimed
4000) with a probe positioned 4 mm posterior to the bregma and 4 mm
lateral from the midline. After 90 minutes MCAO, the filament was with-
drawn to allow for reperfusion. Two hours after onset of occlusion, 60 µg/kg
G-CSF (NEUPOGEN; Amgen Inc.) was infused i.v. over 20 minutes. Infarct
volumes were determined by 2,3,5-triphenyl tetrazolium chloride (TTC)
staining as described previously (9). Two-millimeter sections were cut using
a brain matrix (Harvard Apparatus) and stained with TTC (Sigma-Aldrich)
for 10 minutes at 37°C. Stained sections were scanned on both sides using
a color scanner and infarct areas determined using ImageJ version 1.32j
(http://rsb.info.nih.gov/ij). Edema correction was performed as described
previously (9). For the MCAO model, animals with no or minimal infarcts
(<60 mm3) were excluded from the analysis before unblinding
Combined CCA/distal middle cerebral artery occlusion model. Transient left
combined CCA/middle cerebral artery (CCA/MCA) occlusion model was
achieved as described previously (10). Briefly, animals fasted overnight
were anesthetized with chloral hydrate (0.45 g/kg i.p.). The right femo-
ral vein and artery were cannulated for recording of arterial blood pres-
sure and drug administration. Core body temperature was maintained at
36.5 ± 0.5°C during ischemia and the first hour of reperfusion through
the use of a feed-forward temperature controller. The ipsilateral CCA was
isolated and tagged through a ventral, cervical midline incision. A 0.005-
inch-diameter stainless steel wire (Small Parts Inc.) was placed underneath
the left MCA rostral to the rhinal fissure, proximal to the major bifurca-
tion of the MCA, and distal to the lenticulostriate arteries. The artery was
then lifted, and the wire was rotated clockwise to ensure occlu-
sion. The CCA was next occluded with an atraumatic aneurysm
clip. Cerebral perfusion at the cortical surface, 3 mm distal to
the locus of the MCAO, was measured with a laser Doppler
flowmeter (LDF) (model BPM2; Vasamedic). Only those ani-
mals that displayed a cerebral perfusion of 10–15% of the initial value on
the LDF scale (expressing relative values of cerebral perfusion) were includ-
ed in the study. G-CSF (50 µg/kg) was infused i.v. over 20 minutes starting
60 minutes after induction of ischemia. After 180 minutes of combined
CCA/distal MCA occlusion model, reperfusion was established through
reversal of the occlusion procedure. After 72 hours of reperfusion, ani-
mals were reanesthetized and transcardially perfused with 50 ml of saline.
Perfused isolated brains were transferred into ice-cold PBS for sectioning.
Infarct volumes were determined by TTC staining (see above).
Photothrombotic ischemia model. Male Wistar rats weighing 280 to 320 g
were anesthetized with an intramuscular injection of 100 mg/kg body
weight ketamine hydrochloride (Ketamin 2; Medistar Arzneimittelvertrieb
GmbH) and 8 mg/kg body weight xylazine hydrochloride (Rompun; Bayer).
Anesthesia was maintained with administration of 50 mg/kg body weight
ketamine hydrochloride and 4 mg/kg body weight xylazine hydrochloride
if necessary. A PE-50 polyethylene tube was inserted into the right femoral
artery for continuous monitoring of mean arterial blood pressure and blood
gases. The right femoral vein was cannulated by a PE-50 tube for treatment
infusion. During the experiment, rectal temperature was monitored and
maintained at 37°C by a thermostatically controlled heating pad (FMI
GmbH). Photothrombotic ischemia was induced in the rat parietal cortex
according to the method of Watson et al. (61). Animals were placed in a ste-
reotaxic frame, and the scalp was incised for exposure of the skull surface.
For illumination, a fiber-optic bundle with a 1.5-mm aperture was placed
stereotaxically onto the skull 4 mm posterior to the bregma and 4 mm lat-
eral from the midline. The skull was illuminated with a cold, white light
beam (150 W) for 20 minutes. During the first 2 minutes of illumination,
the dye rose bengal (0.133 ml/kg body weight, 10 mg/ml saline) was injected
i.v. Sham-operated animals underwent the same experimental procedures
as described above without infusion of rose bengal and illumination. After
surgery, the catheters were removed, and the animals were allowed to recov-
er from the anesthesia and given food and water ad libitum. For treatment,
G-CSF increases neurogenesis in the dentate gyrus. (A)
Example of BrdU/NeuN-double-positive cells within the
basal layer of the dentate gyrus (scale bar: 40 µm). The
arrow in A indicates the enlarged double-stained cell in B
(scale bar: 10 µm). (C) DCX in red. (D) BrdU in green. (E)
NeuN in blue. (F) G-CSF increased the number of newly
generated neurons (BrdU+/NeuN+) on the side of the isch-
emic lesion (red bars, ipsilateral + vehicle vs. ipsilateral +
G-CSF; **P < 0.01). Contralateral to the lesion, there was
a trend toward an increase in newly generated neurons
compared with vehicle-treated ischemic animals that was
not statistically significant (blue bars, contralateral + vehicle
vs. contralateral + G-CSF). However, G-CSF increased neu-
rogenesis in sham-operated, nonischemic animals (green
bars, sham + vehicle vs. sham + G-CSF; *P < 0.05). (G)
The total number of BrdU+ cells in the dentate gyrus was
not significantly further increased by G-CSF treatment in the
ischemic animals (red and blue bars), which implies a true
induction of neuronal differentiation by G-CSF in the post-
ischemic brain. In contrast, sham-lesioned animals showed
an elevation of the total number of BrdU+ cells after G-CSF
treatment (green bars; *P < 0.05).
2094? The?Journal?of?Clinical?Investigation http://www.jci.org Volume 115 Number 8 August 2005
ischemic or sham-operated animals were given 15 µg G-CSF/kg body weight
i.v. or vehicle 1 hour after the procedure. Daily repeated i.v. bolus infusions
via the tail vein (G-CSF or vehicle) followed on days 2–5. Four hours after
each tail vein injection of G-CSF or vehicle on days 1–5, dividing cells were
labeled with BrdU by i.p. injections (50 mg/kg/d). We perfused animals 37
days after the last injection in order to histologically assess the amount of
progenitor cells and newly generated neurons.
All animal experiments were performed in accordance with national and
international regulations and were approved by the Regierungspräsidium
Karlsruhe (Karlsruhe, Baden-Württemberg, Germany) and the Animal Wel-
fare Committee of the University of Texas — Houston (Houston, Texas, USA).
All experiments were done in a fully randomized and blinded fashion.
Combined CCA/distal MCA occlusion model. All sensorimotor tests were per-
formed during the light cycle between morning and early afternoon. Ani-
mals were tested at 72 hours just before they were killed for infarct volume
determination. Testing was performed by an investigator blinded to the
experimental groups. A neurological deficit score (0 to 18) was calculated
by combining the scores of the following 4 tests: forelimb placing (both
whisker and forward), foot-fault, and cylinder tests. Tests were done as
described in detail in ref. 62.
Photothrombotic model. In all animals, a battery of behavioral tests was
performed during the light cycle before ischemia after a training period
of 3 days and at 2, 3, 4, 5, and 6 weeks after ischemia by an investigator
blinded to the experimental groups. Tests such as the rotarod, adhesive
tape removal, and beam balance were done as described in detail in ref. 63.
NSS was modified according to ref. 64. Neurological function was graded
on a scale of 0 to 16 (normal score, 0; maximal deficit score, 16). NSS is a
composite of motor, sensory, and reflex tests and includes the beam bal-
ance test (65). In the NSS, 1 score point is awarded for the inability to per-
form the test or for the lack of a tested reflex; thus, the higher the score,
the more severe the injury.
G-CSF and BBB-impermeable BSA (control) were radiolabeled with 131I
(Amersham Biosciences) by the 1,3,4,6-tetrachloro-3α,6α-diphenylgly-
couril (Iodo-Gen; Pierce) method (66) and purified on Sephadex G-25
(Amersham Biosciences) columns. The radiolabeled proteins were injected
via the tail vein of nonischemic female Sprague-Dawley rats (250–300 g).
After 1, 4, and 24 hours following injection of substances, animals were ter-
minally anesthetized with Rompun/Ketanest and perfused carefully with
HBSS, and the whole brain was dissected, blotted dry, and weighed. Blood
was centrifuged to obtain the serum. The radioactivity was measured with
a gamma counter (LB 951G; Berthold Technologies GmbH) along with a
sample of the injection solution to calculate the percent of injected dose
per gram of the tissues.
During deep anesthesia, animals were transcardially perfused with 4%
paraformaldehyde, and brains were removed and either prepared as free-
floating cryosections (40 µm) or embedded in paraffin. For G-CSF or
G-CSF receptor immunohistochemistry, sections of paraffin-embedded
tissues (2 µm) were deparaffinized and microwaved (in citrate buffer at 500
W for 10 minutes). Afterwards, sections were incubated at room tempera-
ture with the respective antisera (1:500; Santa Cruz Biotechnology Inc.)
for 1 hour in a humid chamber. Staining was visualized using the avidin-
biotin complex (ABC) technique with DAB as chromogen (DakoCytoma-
tion). For double immunofluorescence, sections were incubated with the
G-CSF receptor antiserum (1:100), and following incubation with an anti-
rabbit FITC-conjugated secondary antibody (1:200; Dianova), either the
G-CSF (1:100), GFAP, or NeuN antisera (1:100; Chemicon International)
were applied. For detection, the sections were incubated with an appropri-
ate TRITC-conjugated secondary antibody (1:200; Dianova), and nuclear
staining was performed using DAPI. For negative controls, the primary
antiserum was omitted. All double-fluorescence experiments were con-
trolled by parallel single stainings and checked for any fluorescence cross-
talk between detection channels. Double-fluorescence stainings were also
performed with switched chromophores for the secondary antibody.
Human autopsy samples
Formaldehyde-fixed, paraffin-embedded brain tissue samples from a patient
suffering from acute ischemic stroke within the anterior circulation as well
as an age- and sex-matched control were obtained from routine autopsy
cases. Permission for storage and use of human autopsy tissue was obtained
from the Ethikkommision der Medizinischen Fakultät der Universität
Münster in Münster, Germany. Clinical records, autopsy reports, as well
as radiological findings were reviewed in order to determine onset of neu-
rological symptoms and to exclude malignancy, hematological disorders,
sepsis or concomitant neurological disease. The 81-year-old female stroke
patient had died 3 days upon documented onset of neurological symptoms
due to progressive brain edema. The control patient (female, 82 years old)
had died upon acute gastrointestinal bleeding. The intervals between death
and autopsy were 67 hours and 56 hours, respectively. After antigen retrieval
(boiling in 10 mM citrate buffer, pH 6), sections from the ipsilateral and
contralateral cortex (stroke patient) or frontal cortex (control) were stained
using specific antibodies against the G-CSF receptor (1:100; Santa Cruz Bio-
technology Inc.), appropriate secondary antibodies, and the ABC technique
using an automated staining system (TechMate; DakoCytomation).
Neural stem cells were dissociated and plated on poly-l-ornithin/lam-
inin–coated 96-well plates at a density of 30,000 cells/well. After 2 days,
stem cells were washed with PBS (Invitrogen Corp.) (37°C) and fixed with
4% paraformaldehyde for 10 minutes on ice. Then cells were washed with
PBS (4°C) and stored at 4°C. Cells were incubated for 10 minutes in 50
mM glycine in PBS and then washed with PBS. After permeabilization on
ice using 0.2% Triton X-100 (Sigma-Aldrich) in PBS, cells were incubated
with blocking solution (1% BSA in PBS) at room temperature. The G-CSF
receptor antiserum (1:100; Santa Cruz Biotechnology Inc.) and the nestin
antiserum (1:100; BD Transduction Laboratories) were incubated over
night at 4°C. Cells were then washed with 0.1% BSA in PBS and incubated
for 1 hour with the secondary antibodies (anti-rabbit FITC and anti-mouse
TRITC, 1:200; Dianova) at room temperature. Cells were then washed
briefly in 0.1% BSA in PBS and stained with Hoechst 33342 (Invitrogen
Corp.) (1:10,000 in PBS).
In situ hybridization
Riboprobes were generated from PCR-generated templates of cloned
human G-CSF cDNA using T7 (antisense probe) or T3 (sense probe)
polymerase (Roche Diagnostics Corp.). Transcripts were labeled with
rUTP-biotin. Specificity and concentration of transcripts were verified by
denaturing PAGE. Two-micrometer paraffin sections were deparaffinized,
prehybridized (50% formamide; 1% sarcosyl; 0.02% SDS; 5× SSC; 2× wash-
ing reagent from the DIG Wash and Block Buffer Set [Roche Diagnostics
Corp.]), partially digested with proteinase K, washed with 0.1 M glycine/
PBS, and incubated with 500 ng/ml probe overnight at 60°C. Sections were
washed, RNAse A treated (buffer: 5 mM Tris-HCl, pH 8.0; 250 mM NaCl;
0.5 mM EDTA), and incubated for 2 hours with Streptavidin-AP (Roche
Diagnostics Corp.), and stainings were developed using nitro blue tetra-
? The?Journal?of?Clinical?Investigation http://www.jci.org Volume 115 Number 8 August 2005
zolium chloride and 5-bromo-4-chloro-3-indolyl phosphate, toluidine
salt (NBT/BCIP) in detection buffer (100 mM Tris-HCl, pH 9.5; 100 mM
NaCl; 50 mM MgCl2) overnight at room temperature. Sections were coun-
terstained using hemalum and photographed digitally (Olympus IX70).
Sense controls did not yield any specific staining.
Detection of G-CSF mRNA in microdissected cells
Horizontal cryostat brain sections (8 µm) were prepared from mouse brain
and were mounted on frame slides (POL-membrane, 0.9 µm; Leica Micro-
systems). Sections were stained with Alexa 488–conjugated NeuN or GFAP
antibodies (Chemicon International) using a rapid immunohistochemistry
protocol. Identified single neurons or astrocytes were isolated from the
frontal cortex using laser microdissection (Leica). One hundred cells were
pooled in 75 µl RNA lysis buffer (QIAGEN) and stored at room tempera-
ture until further processing. RNA was isolated using the RNeasy Micro
Kit (QIAGEN). RNA quality and quantity were checked by analyzing 1 µl
of RNA on the Agilent 2100 Bioanalyzer using the RNA 6000 Pico Lab-
Chip Kit (Agilent Technologies). T7-RNA polymerase-mediated linear
amplification was performed according to optimized protocols for low-
input RNA amounts. Briefly, after first- and second-strand cDNA synthe-
sis, RNA was transcribed with the T7 MEGAscript Kit (Ambion Inc.) at
37°C for 16 hours. Amplified antisense RNA was purified with the RNeasy
Mini Kit (QIAGEN) and precipitated. First-strand cDNA was synthesized
using random primers, which was followed by second-strand synthe-
sis. PCR was performed for cyclophilin B (mm-cycB1s, TTGCTGCAGC-
CATGGTCAAC; mm-cycB1as, ATTCAGTCTTGGCAGTGCAG, product
length 371 bp), GFAP (mGFAPs, CCCCATCCGCTCAGTCATCTTACC;
mGFAPas, TGTCTTCCCTACCTGCCCACCAAT, product length 280 bp),
and G-CSF (GCSF-790s, GGAGCTCTAAGCTTCTAGATC; GCSF-1154as,
TAGGGACTTCGTTCCTGTGAG, product length 364 bp) under the fol-
lowing conditions: GFAP and cyclophilin were amplified over 30 cycles at
an annealing temperature of 60°C; G-CSF was amplified over 50 cycles at
an annealing temperature of 64°C. As a positive control, a brain-specific
plasmid library was used. Products were visualized by agarose gel electro-
phoresis and ethidium bromide staining.
Primary neuronal cultures
Ten to 12 cortices or hippocampi were dissected from Wistar rat embryos
on E18. The tissue was dissociated using 10 mg/ml trypsin, 5 mg/ml
EDTA/DNase (Roche Diagnostics Corp.) in HBSS (BioWhittaker Molec-
ular Applications). The digestion was stopped using 4 parts neurobasal
medium containing 1× B-27 supplement (Invitrogen Corp.), 0.5 mM
l-glutamine, and 25 µM glutamate. After centrifugation, the cell pellet
was dissolved in 5 ml medium and plated at a density of 250,000 cells per
well of a 24-well-plate on glass coverslips coated with poly-l-lysine (for
immunocytochemistry or Western blot analyses) or into 96-well plates at
5 × 104 cells /well (for cell death assays).
Caspase activity assays
For caspase-3/7 assays, we used the human neuroblastoma cell line
SHSY-5Y or rat primary cortical neurons. Cells were seeded into 96-well
plates (5 × 104 cells /well) for 2 (SHSY-5Y) or 14–21 days (neurons). To
elicit programmed cell death, we treated cells with either the NO donor
NOR3 [(±)E)-4-ethyl-2-[(E)-hydroxyimino]-5-nitro-3-hexenamide] (Sigma-
Aldrich) at 150 µM, 1 µM staurosporine, or 20 µM camptothecin (both
Merck-Calbiochem) for 5 hours with or without recombinant human
(NEUPOGEN; Amgen Inc.) or murine (R&D Systems) G-CSF at 50 ng/ml.
For receptor blocking experiments, primary cortical neurons from the rat
were preincubated for 1 hour with 1 µg/ml anti–G-CSF receptor antibody
(SC 9173; Santa Cruz Biotechnology Inc.) and then treated with 20 µM
camptothecin to induce apoptosis. G-CSF was added to a final concentra-
tion of 50 ng/ml. Incubation was continued for 5 hours, and caspase-3/7
activity was determined by the Caspase-Glo Assay (Promega). For inhibi-
tion of PI3K, the inhibitor LY294002 (Merck-Calbiochem) was added
at 50 µM final concentration 30 minutes prior to cell death stimuli and
G-CSF. Caspase-3/7 activity was determined after 5 hours by the Caspase-
Glo Assay (Promega), and luminescence measured with a plate reader
(Mithras; Berthold Technologies GmbH). Eight to 16 independent data
points were generated for each treatment.
Western blots analyses
For time series pathway analyses, rat primary cortical neurons (21 days in
vitro [DIV]) were treated with G-CSF (NEUPOGEN; Amgen Inc.) and harvest-
ed at 5, 15, 30, and 60 minutes. Experiments were repeated at least twice with
independent preparations of neurons. For determination of PARP cleavage,
neurons (21 DIV) were treated with 150 µM NOR3 with or without G-CSF
(50 ng/ml). Primary neurons were scraped off the plate and washed twice in
ice-cold PBS containing 2.5 mg/ml pepstatin (Sigma-Aldrich) and aprotinin
(1:1,000; Sigma-Aldrich). Pellets were resuspended in 1 volume 2% SDS (40
µl), and 5 µl Benzonase solution (40 µl 100 mM MgCl2 and 9 µl Benzonase;
Roche Diagnostics Corp.) was added. After solubilization, 1 volume PBS was
added and the protein concentration determined (BCA Protein Assay; Pierce).
After denaturing at 95°C for 5 minutes, 100 µg were run on 8% SDS-poly-
acrylamide gels. Proteins were transferred onto nitrocellulose membranes
(Protan BA79; Schleicher & Schuell) using a semi-dry blotting chamber
(Whatman Biometra). Blots were blocked with 5% milk powder in PBS/0.02%
Tween-20, washed 3 times with PBS/0.02% Tween-20, and incubated for 1
hour at room temperature with the primary antibody (anti–cleaved PARP-
antibody, 1:1000 [Cell Signaling Technology]; anti–Bcl-2 antibody, 1:500 [BD
Transduction Laboratories]; anti-STAT3 and anti–phosphorylated STAT3
antibodies, 1:500 [Cell Signaling Technology]; all other antibodies were from
Cell Signaling Technology). After washing, the blots were incubated with the
secondary antibody (anti-rabbit antiserum HRP-coupled or anti-mouse anti-
serum HRP-coupled, 1:4,000; Dianova) for 1 hour at room temperature. Sig-
nals were detected using the SuperSignal chemiluminescence system (Pierce)
and exposed to Hyperfilm-ECL (Amersham Biosciences). Intensities of bands
for phosphorylated Stat3 and Stat3 were quantified on scanned autoradio-
graphs using Windows ImageJ version 1.29.
Quantitative PCR analysis
RNA of brains was isolated using the acidic phenol extraction protocol fol-
lowed by QIAGEN RNeasy Mini Kit purification according to the manu-
facturer’s recommendations. cDNA was synthesized from 5 µg total RNA
using oligo-dT primers and Superscript II Reverse Transcriptase (Invitrogen
Corp.). Quantitative PCR analysis was performed using the LightCycler sys-
tem (Roche Diagnostics Corp.) with SYBR green staining of DNA double
strands. Cycling conditions were as follows: 5 minutes at 95°C, 5 seconds
at 95°C, 10 seconds at 66°C, 30 seconds at 72°C, 10 seconds at 84°C for 50
cycles. Melting curves were determined using the following parameters: 95°C
cooling to 50°C; ramping to 99°C at 0.2°C/second. The following primer
pairs were used: rat G-CSFR-frag-32s, CCATTGTCCATCTTGGGGATC;
rat G-CSFR-frag-265as, CCTGGAAGCTGTTGTTCCATG; G-CSF-345s,
CACAGCGGGCTCTTCCTCTACCAA; G-CSF-862as, AGCAGCGGCAG-
GAATCAATACTCG. The LightCycler PCR analysis was performed using
the SYBR Green master mix, according to the manufacturer’s recommen-
dations (Roche Diagnostics Corp.). Specificity of product was ensured by
melting point analysis and agarose gel electrophoresis. cDNA content of
samples was normalized to the expression level of cyclophilin (primers: cyc5,
ACCCCACCGTGTTCTTCGAC; acyc300, CATTTGCCATGGACAAGATG).
Relative regulation levels were derived after normalization to cyclophilin.
2096? The?Journal?of?Clinical?Investigation http://www.jci.org Volume 115 Number 8 August 2005
Neurogenesis detection in vivo
Progenitor activity and neurogenesis were visualized by immunofluorescence
as previously described (44, 67), but a brief description is given below. The
following antibodies were used: rat anti-BrdU (1:500; Accurate Chemical
& Scientific Corp.), mouse anti-NeuN (1:500, Chemicon International),
goat anti-DCX C-18 (1:500; Santa Cruz Biotechnology Inc.), anti-rat FITC,
anti-goat rhodamine X, anti-mouse Cy5 (Jackson ImmunoResearch Labo-
ratories Inc.). Free-floating sections were treated with 0.6% H2O2 in Tris-
buffered saline (TBS: 0.15 M NaCl, 0.1 M Tris-HCl, pH 7.5) for 30 minutes.
BrdU-labeled nuclei were detected by immunofluorescence after DNA
denaturation: 2 hours incubation in 50% formamide/2× SSC (2× SSC: 0.3
M NaCl, 0.03 M sodium citrate) at 65°C, 5 minutes rinse in 2× SSC, 30
minutes incubation in 2 M HCl at 37°C, and 10 minute rinse in 0.1 M
boric acid, pH 8.5. Thereafter, incubation in TBS/0.1% Triton X-100/3%
normal donkey serum (TBS-TS) for 30 minutes was followed by incubation
with primary antibodies for 48 hours at 4°C. After washing in TBS-TS,
sections were incubated with secondary antibodies for 2 hours, extensively
washed in TBS, and mounted on glass slides. Fluorescence was detected
using a confocal scanning laser microscope (Leica).
A systematic, random counting procedure was used as previously described
(44, 67). Series of every 10th section (400-µm interval) were analyzed. For
the dentate gyrus, all BrdU-positive cells in the granule cell layer were
counted on approximately 6 sections per animal. For colabeling with
neuronal marker NeuN to estimate the percentage of neurons among the
newly generated cells, 100 randomly selected BrdU-positive cells per ani-
mal were analyzed under the confocal microscope. Multiplying the total
number of BrdU-positive cells by the percentage of NeuN/BrdU-double-
positive cells yielded the number of new neurons in the dentate gyrus. For
the lateral ventricle wall, striatum, and cortex, we systematically analyzed 2
sections per animal by counting cells in 25 adjacent frames (250 × 250 µm
each) arranged in an array of 5 × 5 frames starting adjacent to the lesion
site. BrdU-positive, DCX-positive, and NeuN/DCX–double positive cells
were counted, and the density for each cell type was calculated.
Cultivation of adult neural stem cells
Neural stem cells were obtained from the hippocampus or SVZ of 4- to
6-week-old male Wistar rats as described in ref. 68. Briefly, animals were
sacrificed, and brains were dissected and washed in ice-cold Dulbecco’s
PBS (DPBS) containing 4.5 g/l glucose (DPBS/Glc). The hippocampus
and SVZ from 6 animals were dissected, washed in 10 ml DPBS/Glc, and
centrifuged for 5 minutes at 1,600 g at 4°C. Tissue was minced using scis-
sors. Tissue pieces were washed again and centrifuged for 5 minutes at
800 g and the pellet resuspended in 0.01 % (wt/vol) papain, 0.1 % (wt/vol)
Dispase II (Roche Diagnostics) (neutral protease), 0.01 % (wt/vol) DNase I,
and 12.4 mM manganese sulfate in HBSS. Tissue was incubated for 40
minutes at room temperature.
Subsequently, the suspension was centrifuged at 4°C for 5 minutes at
800 g and the pellet washed 3 times in 10 ml DMEM/Ham’s F-12 medium
containing 2 mM l-glutamine, 100 U/ml penicillin/streptomycin. Cells were
then resuspended in 1 ml neurobasal medium containing B27 (Invitrogen
Corp.), 2 mM l-glutamine, 100 U/ml penicillin/streptomycin, 20 ng/ml
EGF, 20 ng/ml bFGF, and 2 µg/ml heparin. Cells were seeded into 6-well
plates at a concentration of 25,000–100,000 cells/ml and incubated at 37°C
in 5% CO2. Two-thirds of the medium volume was changed weekly (68).
Assessment of differentiation markers in vitro
Quantitative PCR. Cultured neurospheres (39 DIV) derived from the SVZ
were stimulated once with the following G-CSF concentrations: 10 ng/ml,
100 ng/ml, and 500 ng/ml. Four days after addition of recombinant
human G-CSF (NEUPOGEN; Amgen Inc.), cells were harvested for the
RNA preparation. Untreated cells served as control. RNA of the G-CSF–
treated and untreated neurospheres of the SVZ was isolated using the
QIAGEN RNeasy Mini Kit according to the manufacturer’s recommenda-
tions. cDNA was synthesized from 2 µg total RNA using oligo-dT primers,
Superscript II Reverse Transcriptase (Invitrogen Corp.). Quantitative PCR
was performed using the LightCycler system (Roche Diagnostics Corp.)
with SYBR green staining of DNA double strands. Cycling conditions
were as follows: nestin and NSE, 3 minutes at 95°C, 5 seconds at 95°C, 10
seconds at 58°C, 30 seconds at 72°C, 10 seconds at 81°C for 50 cycles;
β-III-tubulin, 3 minutes at 95°C, 5 seconds at 95°C, 10 seconds at 65°C, 30
seconds at 72°C, 10 seconds at 87°C for 50 cycles; PLP, 3 minutes at 95°C,
5 seconds at 95°C, 10 seconds at 62°C, 30 seconds at 72°C, 10 seconds at
84°C for 50 cycles; GFAP, 3 minutes at 95°C, 5 seconds at 95°C, 10 seconds
at 60°C, 30 seconds at 72°C, 10 seconds at 81°C for 50 cycles. Melting
curves were determined using the following parameters: 95°C cooling to
50°C; ramping to 99°C at 0.2°C/second. The following primer pairs were
used: rat nestin-plus, AGGAAGAAGCTGCAGCAGAG; rat nestin-minus,
TTCACCTGCTTGGGCTCTAT; rat NSE-plus, GGCAAGGATGCCACTA-
ATGT, rat NSE-minus, AGGGTCAGCAGGAGACTTGA; rat β-III-tub-716s,
CCACCTACGGGGACCTCAACCAC; rat beta III-tub-1022as, GACATGC-
GCCCACGGAAGACG; rat PLP-518s, TCATTCTTTGGAGCGGGTGTG;
rat PLP-927as, TAAGGACGGCAAAGTTGTAAGTGG; rat GFAP3′-1123s,
CCTTTCTTATGCATGTACGGAG; rat GFAP3′-1245as, GTACACTA-
ATACGAAGGCACTC. The LightCycler PCR analysis was performed using
the SYBR Green master mix, according to the manufacturer’s recommen-
dations (Roche Diagnostics Corp.). Specificity of product was ensured by
melting point analysis and agarose gel electrophoresis. cDNA content of
samples was normalized to the expression level of cyclophilin (primers: cyc5,
ACCCCACCGTGTTCTTCGAC; acyc300, CATTTGCCATGGACAAGATG).
Relative regulation levels were derived after normalization to cyclophilin and
comparison with the untreated cells.
Luciferase assay.?To amplify the β-III-tubulin gene promoter (fragment
–450 to +54) (69), rat genomic DNA was used as a template for PCR. The
amplified fragment was inserted into the MluI/XhoI site of the pGL3-Basic
firefly luciferase reporter vector (Promega) to generate the pGL3-p–β-III-
tubulin experimental vector. For DNA transfection, adult neural stem cells
were dissociated and plated on poly-l-ornithin/laminin–coated 96-well
plates at a density of 35,000 cells/well. After 24 hours cultivation, cells were
washed once with 1× DPBS (Invitrogen Corp.). Cotransfection with the
pGL3-p–β-III-tubulin vector (150 ng/well) and a Renilla luciferase construct
(100 ng/well) was carried out with the Lipofectamine method (Invitrogen
Corp.). The pGL3-Basic firefly luciferase reporter vector served as negative
control. The DNA-Lipofectamine 2000 complexes were added to each well
after removal of the DPBS, without addition of neurobasal medium. Fol-
lowing the incubation of transfected cells for 24 hours, cells were stimulat-
ed with various concentrations of G-CSF in neurobasal medium (5 ng/ml,
10 ng/ml, 100 ng/ml) for 48 hours. As a positive control for in vitro differ-
entiation, stem cells were treated by withdrawal of mitogens and addition
of 5% FCS. Using the Dual-Luciferase Reporter Assay System (Promega),
we obtained the ratio of luminescence signals from firefly and Renilla lucif-
erase (Mithras LB 940; Berthold Technologies GmbH).
FACS analysis. For differentiation experiments, adult neural stem cells
were plated in 15 cm2 culture flasks at a density of 4 million cells and were
treated once with 100 ng/ml G-CSF. A single-cell suspension was made by
triturating the neurospheres in 1-ml plastic pipettes and then collected by
centrifugation. After resuspension in 1× PBS, the cells were fixed with 1%
paraformaldehyde. The cells were incubated for 15 minutes on ice, washed
once with 1× PBS, and then permeabilized by resuspension in 0.2% Tween-
?The?Journal?of?Clinical?Investigation http://www.jci.org Volume 115 Number 8 August 2005
20. After an incubation on ice for 15 minutes, FCS was added in a 1:50
dilution for blocking. The cells were incubated for 2 hours on ice with a
MAP2 antibody (1:50; Sigma-Aldrich) and washed 3 times with 0.1% Tween-
20. Following incubation for 30 minutes on ice with a donkey anti-mouse
FITC-conjugated secondary antibody (Dianova), the cells were washed again
3 times with 0.1% Tween-20 and finally resuspended in 1× PBS for FACS
analysis. Flow cytometry of cells was performed on a FACSCalibur (BD).
Data and statistical analysis
The values are presented as mean ± SEM. The 2-tailed Student’s t test
was used to determine significant difference between infarct volumes.
Behavioral measurements were analyzed using the Mann-Whitney rank
sum test or the 2-tailed Student’s t test where appropriate. Area under
the curve was calculated using the trapezoidal algorithm. ANOVA and
post-hoc Duncan test were used to determine the statistical significance
of differences in physiological parameters and neurogenesis. Statistical
analysis was performed with NCSS 2004 software (NCSS). P < 0.05 was
considered statistically significant.
The authors thank Stephan Hennes, Frank Malischewsky, Margit
Wolf, Siena Kiess, Claudia Heuthe, Sandra Bettermann, Andrew
Irving, Katrin Kauf, Rebecca Würz, Robert Aigner, Gisela Eisen-
hardt, Nadine Schramm, Frank Herzog, Ulrike Bolz, Maria
Leiße, and Andrea Esser for excellent technical assistance. Spe-
cial thanks go to Ansgar Brambrink for global ischemia samples
and Moritz Rossner, Dieter Newrzella, and Christine Böhm for
performing laser microdissection and RNA amplification. This
work was supported in part by a grant to H.-G. Kuhn from the
VolkswagenStiftung (I/77 887).
Received for publication October 6, 2004, and accepted in revised
form May 17, 2005.
Address correspondence to: Armin Schneider, Axaron Biosci-
ence AG, Im Neuenheimer Feld 515, 69120 Heidelberg, Ger-
many. Phone: 49-6221-454713; Fax: 49-6221-454713; E-mail:
email@example.com. Or to: Hans-Georg Kuhn, The Arvid Carls-
son Institute for Neuroscience at the Institute for Clinical Neu-
roscience, Sahlgrenska Academy, Gothenburg University, Medici-
naregatan 11, Box 432, 40530 Gothenburg, Sweden. Phone:
46-31-773-3435; Fax: 46-31-773-3401; E-mail: georg.kuhn@neuro.
gu.se. Or to: Wolf-Rüdiger Schäbitz, Universitätsklinikum Mün-
ster, Klinik und Poliklinik für Neurologie, Albert-Schweitzer-Str.
33, 48149 Münster, Germany. Phone: 49-2518-348171; Fax: 49-
2518-348181; E-mail: firstname.lastname@example.org.
C. Krüger and T. Steigleder contributed equally to this work.
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