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Acute Hepatocyte Growth Factor Treatment Induces Long-Term Neuroprotection and Stroke Recovery via Mechanisms Involving Neural Precursor Cell Proliferation and Differentiation


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Hepatocyte growth factor (HGF) is an interesting candidate for acute stroke treatment as shown by continuous infusion or gene delivery protocols. However, little is known about HGF-mediated long-term effects. The present study therefore analyzed long-term effects of an acute intrastriatal HGF treatment (5 μg) after a 45-minute stroke, with regard to brain injury and neurologic recovery. Hepatocyte growth factor induced long-term neuroprotection as assessed by infarct volume and neuronal cell death analysis for as long as 4 weeks after stroke, which was associated with sustained neurologic recovery as evidenced by corner-turn and tight-rope tests. Analyzing underlying mechanisms of HGF-induced sustained neuroprotection, enhanced cell proliferation followed by increased neuronal differentiation of neural precursor cells (NPCs) was observed in the ischemic striatum of HGF-treated mice, which persisted for up to 4 weeks. In line with this, HGF promoted neurosphere formation as well as proliferation of NPC and decreased caspase-3-dependent hypoxic injury in vitro. Preservation of blood-brain barrier integrity 24  hours after stroke was furthermore noticed in animals receiving HGF, which was associated with the inhibition of matrix metalloproteases (MMP)-2 and MMP-9 at 4 and 24  hours, respectively. We suggest that sustained recruitment of proliferating cells together with improved neurovascular remodeling provides an explanation for HGF-induced long-term neuroprotection.
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Acute hepatocyte growth factor treatment induces
long-term neuroprotection and stroke recovery via
mechanisms involving neural precursor cell
proliferation and differentiation
Thorsten R Doeppner
, Britta Kaltwasser
, Ayman ElAli
, Anil Zechariah
Dirk M Hermann
and Mathias Ba
Department of Neurology, University of Goettingen Medical School, Goettingen, Germany;
Department of
Neurology, University of Duisburg-Essen Medical School, Essen, Germany;
DFG Research Center for the
Molecular Physiology of the Brain (CMPB), Goettingen, Germany
Hepatocyte growth factor (HGF) is an interesting candidate for acute stroke treatment as shown
by continuous infusion or gene delivery protocols. However, little is known about HGF-mediated
long-term effects. The present study therefore analyzed long-term effects of an acute intrastriatal
HGF treatment (5 lg) after a 45-minute stroke, with regard to brain injury and neurologic recovery.
Hepatocyte growth factor induced long-term neuroprotection as assessed by infarct volume
and neuronal cell death analysis for as long as 4 weeks after stroke, which was associated
with sustained neurologic recovery as evidenced by corner-turn and tight-rope tests. Analyzing
underlying mechanisms of HGF-induced sustained neuroprotection, enhanced cell proliferation
followed by increased neuronal differentiation of neural precursor cells (NPCs) was observed in
the ischemic striatum of HGF-treated mice, which persisted for up to 4 weeks. In line with this,
HGF promoted neurosphere formation as well as proliferation of NPC and decreased caspase-3-
dependent hypoxic injury in vitro. Preservation of blood–brain barrier integrity 24 hours after stroke
was furthermore noticed in animals receiving HGF, which was associated with the inhibition of
matrix metalloproteases (MMP)-2 and MMP-9 at 4 and 24 hours, respectively. We suggest that
sustained recruitment of proliferating cells together with improved neurovascular remodeling
provides an explanation for HGF-induced long-term neuroprotection.
Journal of Cerebral Blood Flow & Metabolism (2011) 31, 1251 1262; doi:10.1038/jcbfm.2010.211; published online 1 December 2010
Keywords: cerebral ischemia; hepatocyte growth factor; matrix metalloproteases; neural precursor cells;
neurogenesis; stroke
Hepatocyte growth factor (HGF) exhibits unique
features that make it a promising agent for stroke
treatment. In human patients, blood HGF levels in
the acute stroke phase correlate closely with clinical
recovery in the postacute stroke phase, more closely
than other growth factors (Ozaki et al, 2007),
suggesting that HGF induces favorable responses in
the brain tissue that facilitate brain remodeling.
Several studies showed that HGF and its correspond-
ing receptor c-Met, which are present in the mature
brain (Achim et al, 1997; Honda et al, 1995), exert
mitogenic, morphogenic, angiogenic, and antiapop-
totic effects on various kinds of tissues and cells
(Matsumoto and Nakamura, 1996; Nakamura et al,
1989). In these studies, reasons for the beneficial
effects in the brain remained unclear.
Therapeutic approaches using HGF in experimen-
tal stroke research have so far focused on structural
tissue preservation in the acute stroke phase (Date
et al, 2006; Miyazawa et al, 1998; Niimura et al,
2006a,c; Shang et al, 2010; Shimamura et al, 2004).
These studies have mostly been performed on rats
using either continuous infusion or gene delivery
strategies in models of focal or global cerebral
ischemia. Tissue reorganization in the subacute
Received 4 August 2010; revised 18 October 2010; accepted 15
November 2010; published online 1 December 2010
Correspondence: Dr TR Doeppner, Department of Neurology,
University of Duisburg-Essen Medical School, Hufelandstr. 55,
45122 Essen, Germany.
This work was supported by the DFG Research Center for the
Molecular Physiology of the Brain (MB) and DFG (HE3173/2-1,
DMH). HGF for dose finding studies was kindly provided by
Genentech (San Francisco, CA, USA).
Current address: University of Duisburg-Essen, Essen, Germany.
Journal of Cerebral Blood Flow & Metabolism (2011) 31, 1251 1262
2011 ISCBFM All rights reserved 0271-678X/11
stroke phase similarly as long-term functional out-
come after the discontinuation of treatment have not
been examined in these studies. As such, the
question remains open, why HGF is able to induce
persisting neurologic recovery.
In view of their pleiotropic effects on cell prolif-
eration, differentiation, and survival, growth factors
have the potential to influence tissue remodeling in a
profound way far beyond the time point of delivery
(Greenberg and Jin, 2006; Hermann and Zechariah,
2009). Notably, the adult brain possesses its own
source of resting progenitor cells, that is, neural pre-
cursor cells (NPCs) in the subventricular zone (SVZ)
(Ma et al, 2009; Zhao et al, 2008). Upon ischemia,
SVZ-derived NPCs proliferate and migrate toward
the developing brain lesion (Arvidsson et al, 2002;
Yamashita et al, 2006). Whereas many of the new-
born cells undergo degeneration in the postacute
stroke phase (Dayer et al, 2003; Yoo et al, 2008), the
survival of NPCs can greatly be enhanced by growth
factors or antiapoptotic proteins (Doeppner et al,
2009; Wada et al, 2003; Wang et al, 2004).
In view of the promising features of HGF in human
stroke patients, we were interested to see whether
HGF, which initially had been characterized as
organotrophic growth factor outside the brain (Mat-
sumoto and Nakamura, 1996), may induce sustained
neuroprotection when delivered in the acute phase
of a stroke. Furthermore, the question whether or not
tissue survival translates to functional neurologic
recovery was addressed. We therefore conducted
the present experiments, in which we administered
HGF intrastriatally over a time interval of 3 days,
analyzing subsequent processes of tissue remodeling,
with special focus on the endogenous proliferation
and differentiation of NPCs.
Materials and methods
Experimental procedures were performed according to the
European Institutes of Health guidelines for the care and
use of laboratory animals and approved by local autho-
rities. Animals were housed under a regular circadian
rhythm regimen with free access to food and water. In total,
119 (including sham operations) adult male C57/BL6N
mice weighing 23 to 28 g were used. If not stated other-
wise, each experimental group consisted of eight to nine
animals. Mortality rates for animals of experimental groups
including survival periods for up to 7 days were zero for
each experimental condition. For animals surviving 4
weeks after induction of stroke, we observed survival rates
of 88.9% (vehicle treated) and 87.5% (HGF treated). All
experiments were performed in a blinded manner with
regard to the kind of treatment chosen for each animal. For
analysis of postischemic cell proliferation, animals were
intraperitoneally injected with 5-bromo-2-deoxyuridine
(BrdU; Sigma, Steinheim-Taufkirchen, Germany) on days
4 to 6 (7-day survival) or on days 8 to 18 (28-day survival),
with a daily dose of 50 mg/kg body weight.
Induction of Focal Cerebral Ischemia
Induction of focal cerebral ischemia was performed as pre-
viously described (Doeppner et al, 2009). Briefly, animals
were anesthetized (0.8% to 1.5% isoflurane, 30% O
remainder N
O) and rectal temperature was maintained
employing a feedback-controlled heating system. Regional
cerebral blood flow was assessed using a laser Doppler
flowmeter connected to a probe that was attached to the
skull overlying the core region of the middle cerebral artery
territory. Cerebral ischemia was induced by transient
occlusion of the middle cerebral artery using a silicon-
coated filament (180 mm diameter; Doccol, Redlands, CA,
USA), which has been removed after 45 minutes to allow
reperfusion of the middle cerebral artery. Laser Doppler
flowmeter recordings continued for 15 minutes after thread
removal to monitor appropriate reperfusion ( > 80% of
initial regional cerebral blood flow). Sham-operated ani-
mals underwent the same experimental procedure except
for insertion of silicon-coated monofilaments.
Stereotactic Intracerebral Injections
Stereotactic surgeries were performed during reperfusion
under constant anesthesia as described above. The mice
were placed in a stereotactic apparatus (Kopf Instruments,
¨sseldorf, Germany) and the skull was exposed followed
by drilling a hole at 0.4 mm anterior and 1.8mm lateral (left
ischemic hemisphere) from bregma. From these coordi-
nates, intrastriatal injections were made 3.5 mm ventral to
the bregma using a 10-mL Hamilton syringe (Switzerland).
Control mice received 5 mL of 0.1 mol/L phosphate-buffered
saline (PBS) per animal and day as vehicle, whereas treated
animals obtained HGF at doses of 1, 5, or 10 mg on occasion
of each injection, diluted in 5 mL 0.1 mol/L PBS. At the end
of injection, the syringe was kept in place for additional
5 minutes before removal. For experiments with survival
periods of 7 or 28 days, all animals received additional
injections on days 1 to 3 using the same protocol as
described above. For dose-finding studies, HGF was kindly
provided by Genentech (San Francisco, CA, USA), whereas
for the remaining experiments, HGF was purchased from
R&D Systems (Wiesbaden-Nordenstadt, Germany). In the
latter experiments, an HGF dose of 5 mg per animal and day
was used.
Immunohistochemistry and Terminal Transferase
dUTP Nick End Labeling
Animals were intraperitoneally injected with chloralhy-
drate (420 mg/kg body weight) and transcardiacally per-
fused with 4% paraformaldehyde on day 7 or day 28 after
stroke. Brains were removed, postfixed in paraformalde-
hyde, embedded in paraffin, and coronal sections were
prepared. Sections were deparaffinized, boiled in 0.2%
citrate buffer, and incubated with blocking solution. For
analysis of BrdU
cells and Ki-67
cells, sections were
exposed to blocking solution and subsequently stained
with a rat monoclonal anti-BrdU antibody (1:50; 18 hours,
41C; Serotec, Du
¨sseldorf, Germany) or a rabbit monoclonal
Long-term neuroprotection induced by HGF treatment
TR Doeppner et al
Journal of Cerebral Blood Flow & Metabolism (2011) 31, 1251 1262
anti-Ki-67 antibody (1:100; 18 hours, 41C; Abcam,
Cambridge, UK), followed by incubation with a goat anti-
rat Alexa 594 (1:500, 1 hour; Molecular Probes, Darmstadt,
Germany) or a donkey anti-rabbit Alexa Fluor 568 (1:400,
1 hour; Molecular Probes) secondary antibody. For differ-
entiation analysis of BrdU
cells, sections were incubated
with a mouse monoclonal anti-BrdU antibody (1:500;
Roche, Basel, Switzerland), a goat polyclonal antidouble-
cortin antibody (1:50; Santa Cruz Biotechnology, Heidel-
berg, Germany), a rat polyclonal anti-glial fibrillary acidic
protein antibody (1:500; Zymed, Darmstadt, Germany), or a
rat biotin-conjugated antiisolectin B
) antibody (1:100;
Vector, Peterborough, UK) for 18hours at 41C. For further
microglia analysis, colocalization of IB
with major
histocompatibility complex (MHC)-II was performed using
a mouse monoclonal anti-MHC-II antibody (1:200; Santa
Cruz Biotechnology). For double staining against BrdU and
neuronal nuclei (NeuN) or 20,30-cyclic nucleotide 30-
phosphodiesterase, a rat monoclonal anti-BrdU antibody
(see above) and a mouse monoclonal anti-NeuN antibody
(1:200, 18 hours, 41C; Chemicon, Schwalbach, Germany) or
a mouse monoclonal anti-20,30-cyclic nucleotide 30-phos-
phodiesterase antibody (1:400; Chemicon) were used. After
washing, sections were incubated for 1 hour at room
temperature with the following secondary antibodies: goat
anti-mouse Cy-3 (1:400; Dianova, Hamburg, Germany) or
goat anti-rat Alexa 594 (1:400; Dianova) for MHC-II and
BrdU staining, goat anti-rat Alexa 488 (1:250; Molecular
Probes) or donkey anti-goat Alexa 488 (1:250; Molecular
Probes) for glial fibrillary acidic protein or doublecortin
staining, goat anti-mouse Cy-3 (1:100; Jackson Immuno-
Research, Newmarket, UK) for 20,30-cyclic nucleotide 30-
phosphodiesterase staining, and goat anti-mouse Alexa 488
(Molecular Probes) for NeuN (1:400) staining. Terminal
transferase dUTP nick end labeling (TUNEL) staining was
performed incubating sections with proteinase K (7 min-
utes, 371C), followed by exposure to the TdT enzyme
reaction according to the manufacturer’s manual (Roche).
After several washing steps and exposure to TUNEL
blocking solution (20 minutes, room temperature), sections
were stained with a streptavidin-Alexa594-conjugated
secondary antibody (2 hours, room temperature; Molecular
Probes). Quantitative analyses were performed in four
regions of interest within the ischemic striatum located
0.14 mm anterior, 2.5 to 3.25mm ventral, and 1.5 to
2.25 mm lateral from bregma. For each regions of interest,
three sections per animal were evaluated, for which mean
values were computed.
Infarct Volumetry and Determination of Edema
Infarct volume analysis was performed on days 1 and 7
after stroke. Brains were removed and cut into four slices of
2 mm each. Slices were stained with 2,3,5-triphenyltetra-
zolium chloride (2%), and computer-based analysis of
infarct volumes was performed using the software Image J
by subtracting the area of the nonlesioned ipsilateral
hemisphere from that of the contralateral side. Infarct
volume sizes were calculated by integration of the lesioned
areas. Postischemic brain edema was measured also using
Image J software as the increase of ipsilateral hemispheric
volume in comparison to the contralateral hemisphere as
described by Wacker et al (2009).
Analysis of Motor Coordination Deficits
For analysis of poststroke motor coordination deficits,
animals were evaluated using the corner-turn and tight-
rope tests. One day before induction of stroke, all animals
were trained before the beginning of the actual tests
on days 7, 14, 21, and 28 after ischemia. Each test was
performed twice per time point, for which means were
calculated. For the tight-rope test, animals were placed on
a 60-cm long tight rope grasping the string with their
forepaws. Healthy animals grasp the string with four legs
and tail and move to reach a side pole, whereas mice
suffering from ischemia-induced motor deficits cannot lift
their hind legs and eventually fall off onto the cage bedding
placed underneath. Maximum test time was 60 seconds
and the results were scored from 0 (min) to 20 (max)
according to a validated score (Gerlai et al, 2000) depend-
ing on the time animals spent on the rope and whether or
not they reached the platform. Scores were used for
statistical analysis.
For the corner-turn test, animals were placed in a corner
consisting of two vertical boards attached at one side with an
angle of 301. Each mouse was tested for the side chosen over
10 trials per day, that is right or left rearing in the corner.
Whereas healthy animals leave the corner without side
preference, mice suffering from stroke preferentially turn to
the left, nonimpaired body side when leaving the corner. The
laterality index was calculated according to the following
formulae: (number of left turns–number of right turns)/10.
Blood–Brain Barrier Permeability
For this study, animals that were treated with vehicle, HGF
(see above), or the matrix metalloprotease (MMP) inhibitor
BB-1101 (0.75 mg per animal) at the beginning of reperfu-
sion were used. Mice received intravenous injections
of 2% Evans Blue (2 mL/kg) 22 hours after the stroke,
as previously described (Chiba et al, 2008). Two hours
later, animals were killed by transcardiac perfusion with
0.1 mol/L PBS. Brains were removed and separated into
hemispheres. Left hemispheres were weighed, homoge-
nized in 2 mL of 50% trichloroacetic acid, and centrifuged
at 10,000 r.p.m. for 20 minutes. The extracted Evans
Blue dye was then further diluted with ethanol, and
the fluorescence signal was measured with a luminescence
spectrophotometer (excitation at 620 nm, emission at
680 nm). An external standard (62.5 to 500 ng/mL) was
used for the calculation of Evans Blue contents. Evans Blue
contents of left hemisphere tissue was calculated from four
animals as (mg) Evans Blue per (g) tissue.
Zymography of Matrix Metalloproteases
Left hemispheres from ischemic and nonischemic animals
were homogenized in cold lysis buffer (basic buffer)
Long-term neuroprotection induced by HGF treatment
TR Doeppner et al
Journal of Cerebral Blood Flow & Metabolism (2011) 31, 1251– 1262
containing 50 mmol/L Tris-HCl (pH 7.6), 150 mmol/L NaCl,
5 mmol/L CaCl
, 0.05% BRIJ-35, 0.02% NaN3, and 1%
Triton X-100. Homogenates were centrifuged at 41Cat
12,000 gfor 5 minutes and supernatants were thereafter
incubated with a 1:10 volume of gelatin sepharose 4B for
1 hour at 41C with constant shaking. After centrifugation
and resuspension of pellets in elution buffer (basic buffer
containing 10% dimethyl sulfoxide (DMSO) and 20%
volume of lysis buffer), purified samples were analyzed by
zymography. Protein concentrations were determined
by the bicinchoninic acid method (BCA kit, Thermo
Scientific, Bonn, Germany).
Separation of MMP-2 and MMP-9 as pro-form and active
form was performed using Novex Zymogram Gels (Invitro-
gen, Carlsbad, CA, USA) according to the manufacturer’s
instructions. Briefly, samples were incubated in nonredu-
cing sample buffer (0.4 mol/L Tris, pH 6.8, 5% sodium
dodecyl sulfate, 20% glycerol, 0.05% bromophenol blue)
for 10 minutes at room temperature. Thereafter, the
samples were loaded onto 10% sodium dodecyl sulfate
polyacrylamide gel electrophoresis gels containing 0.1%
gelatin. After electrophoresis, the samples were incubated
with 2.5% Triton X-100 twice for 20 minutes, equilibrated
with developing buffer (Novex), and incubated for over
18 hours at 371C. Gels were stained with Coomassie blue
for 30 minutes and destained in washing solution (30%
methanol, 10% acetic acid). White bands on a dark
background indicated zones of digestion corresponding to
the presence of pro-MMPs and activated MMPs on the
basis of their molecular weight. As standards, 0.1 ng of
human pro-MMP-9 and human pro-MMP-2 (Merck Bio-
sciences, Darmstadt, Germany) and 0.01 ng of activated
MMP-9 and activated MMP-2 (Merck Biosciences) were
used. Thereafter, gels were scanned and densitometrically
Preparation and Cultivation of Subventricular
Zone-Derived Neural Precursor Cells
Subventricular zone-derived NPCs were prepared from 11-
to 12-week-old C57/BL6N mice according to a previously
described protocol (Doeppner et al, 2010). Subventricular
zones were microdissected under stereomicroscopic con-
trol and minced into small pieces, mechanically triturated,
and dissociated to single-cell suspension. Cells were
cultured in serum-free basic medium (DMEM-F12; PAA,
Pasching, Austria), supplemented with epidermal growth
factor (2 mg/mL), basic fibroblast growth factor (2 mg/mL),
and penicillin-streptomycin (Invitrogen, Darmstadt,
Germany). Cells were incubated under standard cell
culture conditions and growth factors were added every 2
to 3 days. Cells were passaged every 7 to 10 days and used
for experiments after passage 3 or 4.
Oxygen–Glucose Deprivation
For each experiment, 100,000 NPCs were used (n= 4).
Before induction of oxygen–glucose deprivation (OGD),
cell culture medium was substituted by a crystalloid
solution (‘Thomajodin’ plus 1 mM mannitol; DeltaPharm,
Dortmund, Germany), and the cells were incubated in a
hypoxic chamber (1% O
, and 94% N
) for
45 minutes and reincubated in normal glucose containing
cell culture medium for 24 hours. Control cells were
incubated with 0.1 mol/L PBS 1 hour before OGD, whereas
other cells were treated with recombinant human HGF at a
final concentration of 20 ng/mL. The number of dead cells
was counted using a LIVE/DEAD-Viability-Cytotoxicity-
Assay kit (Lonza, Basel, Switzerland).
Caspase-3 Assay
Caspase-3 activity was measured in cultivated NPCs (n=4
per condition) at 1, 2, 4, and 8 hours after OGD using the
fluorophore substrate Ac-DEVD (aspartic acid-glutamic
acid-valine-aspartic acid)-7-amino-4-methylcoumarin (Bio-
mol, Hamburg, Germany). Cells were incubated either with
HGF (20 ng/mL) or with the caspase-3 inhibitor z-
DEVD.fmk (50 mmol/L, solved in 0.2% DMSO; Biomol) at
the beginning of OGD. Controls were incubated with either
0.1 mol/L PBS or 0.2% DMSO in 0.1 mol/L PBS. At the time
points given, NPCs were complemented with lysis buffer
that contained 25 mmol/L HEPES (pH 7.5), 1 mM EDTA,
10 mM 1,4-dithiothreitol (Roche), 0.1% 3-[(3-cholamido-
propyl)dimethylammonio]-1-propanesulfonate (Sigma-Al-
drich), 10% sucrose, 0.1% Triton X-100, with 10 mg/mL of
pepstatin A, aprotinin, and leupeptin each plus 1 mM PMSF
(all Sigma-Aldrich) as protease inhibitors. Thereafter, the
cells were centrifuged and supernatants were used for
caspase-3 activity measurement. For each measurement,
90 mL of samples were incubated with 10mLofsubstrate
(50 mmol/L final concentration) and fluorescence (355 to
460 nm) was detected at 2-minute intervals for 20 minutes.
The increase in fluorescence was linear between 2 and
16 minutes. Caspase-3 activity was recalculated from t he
slope (fluorescence units per time) and is given as picomoles
of substrate cleaved per milligram of protein per minute
based on a standard curve for 7-amino-4-methylcoumarin
(Biomol). Protein concentration in the supernatant was
determined using the Bradford assay.
All data are given as means±s.d. For comparison between
two groups, the Student’s t-test was used, whereas for a
multigroup comparison, a one-way analysis of variance
followed by the Tukey’s post hoc test was performed.
APvalue of < 0.05 was considered to be statistically
Hepatocyte Growth Factor Induces Neuroprotection in
a Dose-Dependent Manner
Although HGF-induced neuroprotection has been
described before, these studies were mostly per-
formed on rats and included either continuous
release or overexpression of HGF. We therefore first
determined whether or not a single injection of HGF
is sufficient to induce neuroprotection after transient
focal cerebral ischemia in mice (Figure 1A). When
Long-term neuroprotection induced by HGF treatment
TR Doeppner et al
Journal of Cerebral Blood Flow & Metabolism (2011) 31, 1251 1262
1mg of HGF was injected into the ischemic striatum
during early reperfusion, no effect on infarct size
was observed 24 hours after stroke. On the contrary,
HGF at dosages of 5 or 10 mg, respectively, yielded
significant neuroprotection after stroke. In line with
this, ipsilateral edema formation was significantly
reduced in animals treated with 5 or 10 mg of HGF,
but not in animals treated with 1 mg of HGF
(Figure 1B). Hepatocyte growth factor-induced
neuroprotection was not associated with changes in
regional cerebral blood flow (Figure 1C); animals
from all groups exhibited similarly high reperfusion
rates. As no significant difference with regard to
infarct volume and edema formation between ani-
mals treated with 5 or 10 mg of HGF was observed,
further in vivo experiments were performed using
doses of 5 mg only.
Hepatocyte Growth Factor-Induced Neuroprotection
Persists in the Postacute Stroke Phase, and Is
Associated with Ameliorated Motor Coordination
As acute reduction of infarct size and edema
formation (Figure 1) does not imply sustained neuro-
protection or beneficial functional outcome, ische-
mic injury was further analyzed on both day 7 and
day 28 after the stroke (Figure 2). Treatment with
HGF resulted in persistent neuroprotection as
assessed by means of both TUNEL (day 7) and NeuN
(day 28) staining (Figure 2A). We observed 407.6±
54.8 TUNEL
and 701.8±62.2 NeuN
cells per mm
within the periinfarct area of HGF-treated animals
compared with 649.0±80.3 TUNEL
and 533.3±
50.9 NeuN
cells per mm
in control animals. In line
with the sustained neuroprotection, animals treated
with HGF showed reduced postischemic functional
deficits as assessed by both the corner-turn and
the tight-rope tests (Figures 2B and 2C). Although
animals from both vehicle and HGF groups gradually
improved over time, animals treated with HGF
always performed better than vehicle-treated ani-
mals, thereby almost approximating test performance
of healthy sham-operated animals.
Hepatocyte Growth Factor Stabilizes Blood–Brain
Barrier Integrity via Inactivation of Matrix
As development of acute infarct injury involves
enhanced blood–brain barrier (BBB) permeability,
we analyzed HGF-mediated effects on Evans Blue
extravasation 24 hours after induction of stroke.
Whereas control animals showed distinct Evans
Blue extravasation, animals treated with HGF dis-
played significantly reduced extravasation of the dye
(Figure 3A). Similarly, treatment with BB-1101
(0.75 mg per animal), a broad inhibitor of MMPs that
are critically involved in BBB breakdown, also
resulted in reduced Evans Blue extravasation.
Whereas HGF treatment lead to subacute neuropro-
tection as suggested by decreased infarct volumes
on day 7, animals treated with BB-1101 did not benefit
from this therapy. As such, infarct volume was not
influenced by BB-1101 (Figure 3B). Matrix metallo-
proteases gelatin zymography (Figures 3C–3E) re-
vealed activation of MMP-2 in vehicle-treated control
Figure 1 Hepatocyte growth factor (HGF) reduces postischemic
injury and edema formation in a dose-dependent manner.
Hepatocyte growth factor was injected at different dosages into
the left ischemic striatum at the beginning of reperfusion
followed by infarct analysis 24 hours after stroke (A). Ipsilateral
edema formation at 24 hours after stroke is given as relative
increase of ipsilateral hemisphere volume as compared with
the contralateral nonischemic hemisphere (B). Note that
HGF-induced neuroprotection was not associated with regional
cerebral blood flow changes (C). Data (n= 6 animals per group)
are presented as means±s.d. *Significantly different from
Long-term neuroprotection induced by HGF treatment
TR Doeppner et al
Journal of Cerebral Blood Flow & Metabolism (2011) 31, 1251– 1262
animals 4 hours after the stroke, which was not found
in sham-operated animals. Both HGF and BB-1101
treatments reduced MMP-2 activation without chan-
ging levels of inactive pro-MMP-2. Activation of
MMP-2 was, however, only transient, decreasing
below levels of detection within 24 hours after stroke.
On the other hand, active MMP-9 was only detected as
late as 24 hours after stroke. Matrix metalloprotease-9
but not pro-MMP-9 activity was again reduced by HGF
and BB-1101.
Hepatocyte Growth Factor Enhances Postischemic Cell
To further elucidate the mechanisms underlying
the sustained neuroprotection and recovery induced
by HGF, cell proliferation was analyzed using
the thymidine analog BrdU on day 7 and day 28
after stroke (Figure 4A). Although cell proliferation
declined over time within the ischemic striatum of
animals treated with either HGF or vehicle, the
number of BrdU
cells in animals treated with HGF
was always higher than in vehicle-treated controls.
As BrdU labeling does not exclusively reflect current
cell proliferation, analysis of Ki-67
cells was
additionally performed at the time points given
(Figure 4B). Similarly, HGF treatment resulted in
enhanced numbers of Ki-67
cells for up to 4 weeks
after induction of stroke, demonstrating that HGF
enhances postischemic endogenous cell proliferation.
Endogenous Poststroke Neurogenesis Is Enhanced
After Hepatocyte Growth Factor Treatment
Differentiation analysis of BrdU
cells revealed high
relative amounts of BrdU
cells expressing the
microglial marker IB
on day 7 after stroke, suggest-
ing that a majority of proliferating cells rather reflect
microglia than newborn neural cells (Figure 5A).
There was, however, no significant difference be-
tween animals treated with either HGF or vehicle.
With regard to the state of activation of IB
glial cells, double staining against IB
, and MHC-II
on day 7 revealed no difference between vehicle and
HGF group. We observed 93.1%±12.5% in the
vehicle and 89.6%±7.5% of colocalizations in the
HGF group, representing a high state of activation of
microglial cells. Regarding neural differentiation of
cells, animals treated with HGF showed
significantly higher numbers of BrdU
cells expres-
sing the immature neural marker nestin than con-
trols, albeit cell numbers declined over time in both
experimental groups (Figure 5B). On the contrary, the
number of BrdU
cells expressing glial fibrillary
acidic protein gradually increased over time in
animals treated with both HGF or vehicle (Figure
5C). There was, however, no significant difference
between these two groups. Likewise, oligodendro-
glial differentiation of BrdU
cells was not affected
by HGF (Figure 5D). Interestingly, long-term neuro-
nal differentiation was enhanced in animals treated
with HGF compared with vehicle-treated mice
(Figures 5E–5H). Hepatocyte growth factor treatment
resulted in increased numbers of BrdU
expressing either doublecortin or NeuN at day 28
after stroke when compared with control animals.
Whereas no mature neuronal differentiation of
newborn proliferating cells was observed on day 7
Figure 2 Sustained neuroprotection and amelioration of func-
tional neurologic deficits induced by hepatocyte growth factor
(HGF). Treatment with HGF significantly reduced the number of
terminal transferase dUTP nick end labeling (TUNEL
) cells on
day 7 after stroke (A) and significantly increased the density of
neurons on day 28 (A). Long-term neuroprotection by
HGF was accompanied by enhanced motor coordination
performance in both the corner-turn (B) and the tight-rope (C)
tests. Data are presented as means±s.d. *Significantly different
from vehicle. NeuN, neuronal nuclei.
Long-term neuroprotection induced by HGF treatment
TR Doeppner et al
Journal of Cerebral Blood Flow & Metabolism (2011) 31, 1251 1262
(data not shown), the number of BrdU
expressing NeuN was still low on day 28 in both
experimental groups.
Hepatocyte Growth Factor Stimulates Neural
Precursor Cell Proliferation In Vitro and Protects
Neural Precursor Cells Against Hypoxic Injury
As enhanced cell proliferation (Figure 4) can be
either due to stimulation of cell division itself or due
to the protection of proliferating cells, we analyzed
NPC proliferation under physiological conditions
in vitro. When NPCs were cultivated under standard
cell culture conditions, neurosphere formation was
observed in NPCs treated with either vehicle or
HGF (Figures 6A and 6B). However, incubation of
NPCs with HGF resulted in significantly higher
numbers of neurospheres as compared with control
NPCs (Figure 6C). Likewise, treatment of NPCs with
HGF resulted in significantly enhanced numbers of
NPCs incorporating BrdU (Figure 6D), suggesting
that HGF stimulates proliferation of SVZ-derived
NPCs in vitro. Apart from HGF-mediated stimulation
of NPC proliferation, HGF also preserved cultivated
NPC from injury when exposed to 45 minutes of OGD
followed by subsequent reexposure to oxygen and
glucose under standard cell culture conditions
Figure 3 Hepatocyte growth factor (HGF) reduces blood–brain barrier (BBB) permeability and inhibits matrix metalloproteases
(MMP). Blood–brain barrier permeability (A) was assessed 24 hours af ter stroke by means of Evans Blue extravasation (n=4
animals per group). Infarct volumes (B) were analyzed on day 7 after stroke using 2,3,5-triphenyltetrazolium chloride (TTC)
stainings. (C) Gelatin zymography of brain homogenates was performed at 4 or 24 hours after stroke (n= 4 animals per group).
Animals were treated at the beginning of reperfusion. Control animals underwent sham surgery without further treatment. Human
pro-MMPs (0.1 ng) and activated MMPs (0.01 ng) served as standards. (D,E) Densitometric analyses of activated MMP-2 (D) and
activated MMP-9 (E) from gelatin zymography at 4 or 24 hours after stroke, respectively. Data are presented as means±s.d.
*Significantly different from vehicle.
Long-term neuroprotection induced by HGF treatment
TR Doeppner et al
Journal of Cerebral Blood Flow & Metabolism (2011) 31, 1251– 1262
(Figure 6E). Whereas control cells developed promi-
nent and distinct cell injury, NPCs incubated with
HGF either 4 hours before OGD or at the beginning of
OGD showed significantly enhanced survival (Figure
6E). Furthermore, HGF-induced neuroprotection of
NPCs in vitro was associated with attenuated
caspase-3 activation. As such, significantly reduced
caspase-3 activity was observed for up to 8 hours
after OGD in cells that were treated with HGF. As a
matter of fact, caspase-3 activities in HGF-treated
cells were as low as caspase-3 activities from NPCs
treated with z-DEVD.fmk, a well-known inhibitor of
caspase-3. These data suggest that HGF promoted the
proliferation and survival of NPCs via a mechanism
involving caspase-3.
The present study reports that an acute HGF delivery
in the first 3 days after stroke induces long-term
neuroprotection associated with enhanced motor
coordination recovery. Enhanced proliferation and
differentiation of SVZ-derived NPCs were noticed in
HGF-treated mice, which based on in vitro studies
were a consequence of both enhanced cell division
and increased resistance against hypoxic injury.
Blood–brain barrier integrity was also enhanced
and MMP-activation inhibited, pointing toward a
profound remodeling of the brain that clearly out-
lasted the time window of HGF delivery. Our data
exemplify that HGF induces potent restorative
changes in ischemic brain tissue. This might explain
the previously reported association of HGF values
measured in the blood of stroke patients early after
stroke with postacute recovery.
Although HGF-induced neuroprotection has pre-
viously been described in rats using continuous
infusion or gene delivery strategies in models of focal
or global cerebral ischemia (Date et al, 2006; Niimura
et al, 2006b; Shang et al, 2010; Shimamura et al,
2006), we for the first time report that HGF is also
protective in mice. Different to earlier studies that
focused on neuroprotection in the acute injury
phase, we for the first time evaluated effects of
HGF after the discontinuation of treatment, showing
that the promotion of neuronal survival is sustained
over as long as 4 weeks, and that it goes along with
functional neurologic recovery. In dose-finding stu-
dies, we showed that doses of 5 to 10 mg per animal
and day are needed to induce neuroprotection,
whereas a dose of 1 mg per day was ineffective.
Sustained neuroprotection has previously also been
shown for other growth factors with pleiotropic
action, namely for vascular endothelial growth factor
(Sun et al, 2003; Wang et al, 2006) and granulocyte-
colony stimulating factor (Shyu et al, 2004). Com-
pared with the huge number of studies analyzing
effects of various growth factors on tissue survival in
rodent models of ischemic stroke, the number of
studies analyzing long-term structural and functional
effects in the postacute stroke phase is still small.
The lack of studies with clinically relevant struc-
tural and functional end points may represent one
reason for the failure of clinical neuroprotection
trials in the past.
Growth factors have multiple effects on cellular
growth, proliferation, differentiation, and survival,
which may help stimulate neurologic recovery and
promote remodeling, once a stroke has occurred
(Greenberg and Jin, 2006). This explains why growth
factors may induce beneficial effects that outlast the
termination of treatment. Indeed, HGF induced a
long-lasting proliferation response of endogenous
NPCs, which persisted until the end of the experi-
ments at 28 days after stroke. Although HGF has been
shown to enhance neuronal differentiation of em-
bryonic stem cells in vitro before (Kato et al, 2004;
Kokuzawa et al, 2003), HGF-induced effects on
postischemic proliferation of NPCs have not been
studied until now. The propagated proliferation of
NPCs represents a reasonable explanation of the
Figure 4 Hepatocyte growth factor (HGF) promotes post-
ischemic cell proliferation. Postischemic treatment with HGF
resulted in increased cell proliferation as assessed by 5-bromo-
2-deoxyuridine (BrdU) (A) and Ki-67 (B) staining. Cell prolifera-
tion was increased at both time points analyzed. Note a
constant decline of proliferating cells over time in vehicle- and
HGF-treated animals. Data are presented as means±s.d.
*Significantly different from vehicle.
Long-term neuroprotection induced by HGF treatment
TR Doeppner et al
Journal of Cerebral Blood Flow & Metabolism (2011) 31, 1251 1262
sustained action of HGF. Interestingly, both vascular
endothelial growth factor (Sun et al, 2003; Wang
et al, 2006) and granulocyte-colony stimulating
factor (Shyu et al, 2004) promote neurogenesis. In
our study, the majority of NPCs maintained at a
largely undifferentiated state, expressing markers of
immature neural cells. Nonetheless, the rate of
differentiation into mostly immature neurons, astro-
glia, and oligodendroglia was also increased. It is
well conceivable that these cells act in a bystander
manner (Bacigaluppi et al, 2008), orchestrating
restorative responses of the ischemic brain tissue.
Hepatocyte growth factor-mediated stimulation of
postischemic endogenous cell proliferation and
neurogenesis can result from both neuroprotection
of newborn cells due to antiapoptotic properties of
HGF or due to induction of cell proliferation itself.
We therefore analyzed the effects of HGF treatment
on SVZ-derived NPCs in vitro. Interestingly, HGF did
not only induce neurosphere formation and cell
proliferation under physiological conditions, but
also resulted in acute protection of NPCs against
hypoxic injury in a caspase-3-associated mechanism.
Like cerebral ischemia (Mehta et al, 2007), hypoxic
injury comprises a set of different mechanisms
and cellular pathways such as activation of p38 or
JNK. Hepatocyte growth factor-mediated inhibition
of caspase-3 activation in cultured NPCs can there-
fore only be regarded as one possible mechanism
by which HGF induces neuroprotection against
hypoxic injury. Although few studies reported an
HGF-mediated induction of cell proliferation and
neuronal differentiation of embryonic-derived stem
cells in vitro before (Kato et al, 2004; Kokuzawa et al,
2003), only one recent study observed an enhanced
proliferation of cultured SVZ-derived NPCs after
HGF treatment (Nicoleau et al, 2009). Characteristics
of SVZ-derived NPCs, however, also depend on the
species and the age of animals (Baker et al, 2005; Van
Kampen et al, 2004), and the aforementioned study
analyzed HGF-mediated effects on NPCs derived
from rats or mice that were either newborn or adult.
Taken into account that cell proliferation of SVZ-
derived NPCs from mice gradually declines over time
with aging animals (own unpublished observation),
these results cannot be transferred to our study.
According to our in vitro studies, stimulation of
postischemic neurogenesis in vivo by HGF might be a
consequence of both induction of NPC proliferation
and protection of newborn cells within an inflam-
matory and proapoptotic postischemic milieu.
Among different pathophysiological mechanisms
that lead to acute ischemic injury, breakdown of the
BBB is one key factor. In our study, HGF treatment
resulted in reduced extravasation of Blue Evans and
reduced brain edema formation compared with
vehicle control animals. Although HGF-mediated
poststroke modulation of the BBB and the expression
of tight junction proteins has been described before
after continuous HGF infusion in rats (Date et al,
2004, 2006), effects after single bolus injections have
not yet been studied. One has to keep in mind,
Figure 5 Hepatocyte growth factor (HGF) promotes neural differentiation. Fate of differentiation of 5-bromo-2-deoxyuridine (BrdU
proliferating cells was analyzed using the microglial marker isolectin B
)(A), the neural marker nestin (B), the astroglial marker
glial fibrillary acidic protein (GFAP) (C), the oligodendroglial marker 20,30-cyclic nucleotide 30-phosphodiesterase (CNPase) (D), the
immature neuronal marker doublecortin (Dcx) (E,F), and the marker for mature neuronal cells NeuN (G,H). Arrows indicate
colocalization of BrdU
cells (red) with the marker in question (green), that is Dcx (F) or NeuN (H). Scale bars: 40 mm(F) and 20 mm
(H). Data are presented as means±s.d. *Significantly different from vehicle.
Long-term neuroprotection induced by HGF treatment
TR Doeppner et al
Journal of Cerebral Blood Flow & Metabolism (2011) 31, 1251– 1262
however, that a 45-minute stroke in mice as chosen
for the present study might only induce mild BBB
breakdown in comparison to extended ischemia
durations. As activation of MMP is critically
involved in BBB breakdown, we further analyzed
the activation of both MMP-2 and MMP-9 within
24 hours after stroke onset. We found acute activation
of MMP-2 and MMP-9 in control animals after 4 or
24 hours, respectively, which is in line with previous
reports (Candelario-Jalil et al, 2009). Interestingly,
inhibition of MMP activation by HGF was as
effective as treatment with BB-1101, a well-known
unspecific MMP inhibitor. Whereas both HGF and
BB-1101 significantly stabilized BBB integrity, only
HGF yielded neuroprotection. These results confirm
previous studies, indicating that MMP inhibition
does not influence ischemic injury in the long run,
despite an early attenuation of brain damage (Rosen-
berg et al, 1998; Sood et al, 2008).
In conclusion, the present study shows that acute
HGF treatment induces long-term neuroprotection
lasting beyond the discontinuation of treatment,
translating into functional neurologic recovery in
the postacute stroke phase. As histopathological
correlate of its perpetuated action, an enhanced
proliferation and differentiation of SVZ-derived
NPCs was noticed, which again outlasted the dura-
tion of HGF treatment and therefore may have
orchestrated the remodeling of the brain tissue.
Further insights are urgently needed into denomi-
nators of successful and nonsuccessful brain recov-
ery, so that therapies may be tailored to improve
functional restitution.
Disclosure/conflict of interest
The authors declare no conflict of interest.
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... Existing literature supports that MET signaling is neurotrophic and neuroprotective in multiple neurodegenerative mouse models ranging from multiple sclerosis (Bai et al., 2012;Benkhoucha et al., 2014;Matsumoto et al., 2014), Parkinson's disease (Koike et al., 2006), to ALS (Genestine et al., 2011). HGF has also been shown to confer neuroprotection during stroke (Doeppner et al., 2011), ischemia (Shibuki et al., 2002), and Aβ-induced cognitive impairment in mice (Takeuchi et al., 2008). Moreover, the HGF-MET duo modifies central and peripheral immune functions (Benkhoucha et al., 2010), which are emerging as the key regulators of AD pathogenesis (Labzin et al., 2018;Ising et al., 2019;Gate et al., 2020). ...
... The early reduction in MET protein in 5XFAD mice, together with reported neuroprotective role of HGF/MET in animal models (Doeppner et al., 2011), suggests that MET could be a protective factor in AD. If this is the case, ablation of MET signaling, which could be achieved through the cre-loxP technology, may aggravate neuropathology in the 5XFAD mice. ...
Full-text available
Alzheimer's disease (AD) is a neurodegenerative disorder strongly associates with aging. While amyloid plagues and neurofibrillary tangles are pathological hallmarks of AD, recent evidence suggests synaptic dysfunction and physical loss may be the key mechanisms that determine the clinical syndrome and dementia onset. Currently, no effective therapy prevents neuropathological changes and cognitive decline. Neurotrophic factors and their receptors represent novel therapeutic targets to treat AD and dementia. Recent clinical literature revealed that MET receptor tyrosine kinase protein is reduced in AD patient's brain. Activation of MET by its ligand hepatocyte growth factor (HGF) initiates pleiotropic signaling in the developing brain that promotes neurogenesis, survival, synaptogenesis, and plasticity. We hypothesize that if reduced MET signaling plays a role in AD pathogenesis, this might be reflected in the AD mouse models and as such provides opportunities for mechanistic studies on the role of HGF/MET in AD. Examining the 5XFAD mouse model revealed that MET protein exhibits age-dependent progressive reduction prior to overt neuronal pathology, which cannot be explained by indiscriminate loss of total synaptic proteins. In addition, genetic ablation of MET protein in cortical excitatory neurons exacerbates amyloid-related neuropathology in 5XFAD mice. We further found that HGF enhances prefrontal layer 5 neuron synaptic plasticity measured by long-term potentiation (LTP). However, the degree of LTP enhancement is significantly reduced in 5XFAD mice brain slices. Taken together, our study revealed that early reduction of HGF/MET signaling may contribute to the synaptic pathology observed in AD.
... Shimamura et al. found HGF alleviated brain ischemic injury without exacerbating cerebral edema through promoting angiogenesis and inhibiting neuronal apoptosis [23]. Moreover, HGF could promote neural stem cell proliferation and self-renewal, and HGF could enhance neurogenesis by stimulating the proliferation of neural precursor cells and protecting them against hypoxic injury [29,30]. ...
... For example, after HIBD, the ultrastructure of the blood-brain barrier changes, leading to the development of brain edema [41]. Doeppner et al. found that HGF stabilized the integrity of the BBB by inactivating matrix metalloproteases [30]. All of the findings indicate that HGF is a potential candidate for HIBD treatment. ...
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Objectives Hypoxic–ischemic brain damage (HIBD) is a major cause of brain injury in neonates. Bone marrow mesenchymal stem cells (BMSCs) show therapeutic potential for HIBD, and genetic modification may enhance their neuroprotective effects. The goal of this study was to investigate the neuroprotective effects of hepatocyte growth factor (HGF)-overexpressing BMSCs (BMSCs-HGF) against HIBD and their underlying mechanisms. Methods BMSCs were transfected with HGF using adenoviral vectors. HIBD models were established and then BMSCs were transplanted into the brains of HIBD rats via intraventricular injection. 2,3,5-Triphenyltetrazolium chloride (TTC) staining was used to measure cerebral infarction volumes. In vitro , primary cultured cortical neurons were co-cultured with BMSCs in a Transwell plate system. Oxygen–glucose deprivation (OGD) was applied to imitate hypoxic–ischemic insult, and PD98059 was added to the culture medium to block the phosphorylation of extracellular signal-regulated kinase (ERK). Cell apoptosis was determined using TUNEL staining. The expression of HGF was measured by immunofluorescence, real-time quantitative PCR (RT-qPCR), and western blots. The expression of phosphorylated ERK (p-ERK) and B-cell lymphoma-2 (Bcl-2) was measured by western blots. Results HGF-gene transfection promoted BMSC proliferation. Moreover, BMSCs-HGF decreased HIBD-induced cerebral infarction volumes and enhanced the protective effects of the BMSCs against HIBD. BMSCs-HGF also increased expression of HGF, p-ERK, and Bcl-2 in brain tissues. In vitro , BMSC-HGF protected neurons against OGD-induced apoptosis. Inhibition of ERK phosphorylation abolished the neuroprotective effect of BMSCs-HGF against OGD. Conclusions BMSCs-HGF is a potential treatment for HIBD and that the ERK/Bcl-2 pathway is involved in the underlying neuroprotective mechanism.
... IS significantly induced endogenous neurogenesis in the dentate gyrus of the hippocampus. However, newborn neurons are difficult to differentiate into mature neurons (Arvidsson et al., 2002;Doeppner et al., 2011). Quercetin maintains isocitrate dehydrogenase levels in MCAO animal models and helps to preserve neuronal cell energy production, thereby reducing IS-induced neuronal cell damage . ...
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Ischemic stroke (IS) is characterized by high recurrence and disability; however, its therapies are very limited. As one of the effective methods of treating acute attacks of IS, intravenous thrombolysis has a clear time window. Quercetin, a flavonoid widely found in vegetables and fruits, inhibits immune cells from secreting inflammatory cytokines, thereby reducing platelet aggregation and limiting inflammatory thrombosis. In pre-clinical studies, it has been shown to exhibit neuroprotective effects in patients with ischemic brain injury. However, its specific mechanism of action remains unknown. Therefore, this review aims to use published data to elucidate the potential value of quercetin in patients with ischemic brain injury. This article also reviews the plant sources, pharmacological effects, and metabolic processes of quercetin in vivo , thus focusing on its mechanism in inhibiting immune cell activation and inflammatory thrombosis as well as promoting neuroprotection against ischemic brain injury.
... The recovery of neurological function after cerebral ischemia mainly depends on the migration of newly formed neurons to severe ischemic lesions, such as the striatum and granular layer of the hippocampus, to replace necrotic neurons. However, only a very small portion of newly generated neurons can differentiate into mature neurons (2,3). In addition to neurogenesis, the recovery of neurological function after a stroke depends on the neuroplasticity of the established networks in the ipsilateral tissue, including axonal sprouting, dendritic remodeling, and synapse strengthening (4,5). ...
Ischemic stroke can induce neurogenesis. However, most stroke-generated newborn neurons cannot survive. It has been shown that MR-409, a potent synthetic agonistic analog of growth hormone–releasing hormone (GHRH), can protect against some life-threatening pathological conditions by promoting cell proliferation and survival. The present study shows that long-term treatment with MR-409 (5 or 10 μg/mouse/d) by subcutaneous (s.c.) injection significantly reduces the mortality, ischemic insult, and hippocampal atrophy, and improves neurological functional recovery in mice operated on for transient middle cerebral artery occlusion (tMCAO). Besides, MR-409 can stimulate endogenous neurogenesis and improve the tMCAO-induced loss of neuroplasticity. MR-409 also enhances the proliferation and inhibits apoptosis of neural stem cells treated with oxygen and glucose deprivation–reperfusion. The neuroprotective effects of MR-409 are closely related to the activation of AKT/CREB and BDNF/TrkB pathways. In conclusion, the present study demonstrates that GHRH agonist MR-409 has remarkable neuroprotective effects through enhancing endogenous neurogenesis in cerebral ischemic mice.
... recorded in Supplementary Table 1. The induction of transient focal cerebral ischemia in male C57BL/6J mice aged 10 weeks (Janvier Labs, Le Genest-Saint-Isle, France) was obtained using the MCA occlusion model as previously described [63]. Only male mice were studied in order to avoid the interference of the hormonal disturbances of female mice after MCAO surgery. ...
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Systemic transplantation of oxygen−glucose deprivation (OGD)-preconditioned primary microglia enhances neurological recovery in rodent stroke models, albeit the underlying mechanisms have not been sufficiently addressed. Herein, we analyzed whether or not extracellular vesicles (EVs) derived from such microglia are the biological mediators of these observations and which signaling pathways are involved in the process. Exposing bEnd.3 endothelial cells (ECs) and primary cortical neurons to OGD, the impact of EVs from OGD-preconditioned microglia on angiogenesis and neuronal apoptosis by the tube formation assay and TUNEL staining was assessed. Under these conditions, EV treatment stimulated both angiogenesis and tube formation in ECs and repressed neuronal cell injury. Characterizing microglia EVs by means of Western blot analysis and other techniques revealed these EVs to be rich in TGF-β1. The latter turned out to be a key compound for the therapeutic potential of microglia EVs, affecting the Smad2/3 pathway in both ECs and neurons. EV infusion in stroke mice confirmed the aforementioned in vitro results, demonstrating an activation of the TGF-β/Smad2/3 signaling pathway within the ischemic brain. Furthermore, enriched TGF-β1 in EVs secreted from OGD-preconditioned microglia stimulated M2 polarization of residing microglia within the ischemic cerebral environment, which may contribute to a regulation of an early inflammatory response in postischemic hemispheres. These observations are not only interesting from the mechanistic point of view but have an immediate therapeutic implication as well, since stroke mice treated with such EVs displayed a better functional recovery in the behavioral test analyses. Hence, the present findings suggest a new way of action of EVs derived from OGD-preconditioned microglia by regulating the TGF-β/Smad2/3 pathway in order to promote tissue regeneration and neurological recovery in stroke mice.
... On the other hand, HGF was described as a neuroprotector and angiogenesis promoter after cerebrovascular accidents [9]. It was found that HGF levels in the acute stroke phase correlate closely with clinical recovery in the post-acute stroke phase, by inducing long-term neuroprotection lasting beyond the discontinuation of treatment, translating into enhanced motor coordination recovery suggesting that HGF induces favorable responses in the brain tissue that facilitate brain remodeling, and these unique features make it a promising agent for stroke treatment [10,11]. ...
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Background Hepatocyte growth factor (HGF) has an obvious pathological role in atherosclerosis and plaque instability leading to an acute ischemic stroke; however, its beneficial role in stroke recovery is still restricted to experimental studies. The aim of the current study was to investigate the association between HGF and carotid atherosclerosis and evaluate its value as a prognostic marker of ischemic stroke and its role in stroke recovery. Results This case–control study was done on 100 patients with first time anterior circulation ischemic stroke, subjected to clinical and laboratory evaluation of atherosclerosis risk factors. Brain imaging, cardiac work-up and ultrasonographic assessment of carotid atherosclerosis (using intimal medial thickness and plaque score) were all done. Clinical evaluation of initial stroke severity, using National Institutes of Health Stroke Scale (NIHSS), and stroke outcome after 3 m, using Modified Rankin Scale (MRS), was performed. Measurement of HGF serum concentration was done to all stroke patients within 24 h of stroke onset and compared to results of 100 matched healthy subjects aged more than 50 years. HGF was significantly higher in stroke patients than healthy controls and in atherothrombotic than cardioembolic stroke group and its level was significantly correlated with atherosclerosis risk factors, degree of carotid atherosclerosis and better stroke outcome; however, it was not significantly correlated with initial stroke severity. Conclusion HGF is strongly associated with carotid atherosclerosis and other atherosclerosis risk factors and subsequent atherothrombotic stroke. Also, it can be used as a good prognostic marker in atherothrombotic stroke suggesting its role in stroke recovery but more studies are needed to explore this beneficial role as well as its therapeutic potentials in ischemic stroke patients.
... BBB integrity was evaluated by Evans blue extravasation, which was performed as previously described (Doeppner et al., 2011;Radu and Chernoff, 2013). Briefly, 100 µl of 2% Evans Blue dye (Sigma-Aldrich, Darmstadt, Germany) was administered via the femoral vein 2 h before sacrifice. ...
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Inhibition of fatty acid synthesis (FAS) stimulates tumor cell death and reduces angiogenesis. When SH-SY5Y cells or primary neurons are exposed to hypoxia only, inhibition of FAS yields significantly enhanced cell injury. The pathophysiology of stroke, however, is not only restricted to hypoxia but also includes reoxygenation injury. Hence, an oxygen-glucose-deprivation (OGD) model with subsequent reoxygenation in both SH-SY5Y cells and primary neurons as well as a murine stroke model were used herein in order to study the role of FAS inhibition and its underlying mechanisms. SH-SY5Y cells and cortical neurons exposed to 10 h of OGD and 24 h of reoxygenation displayed prominent cell death when treated with the Acetyl-CoA carboxylase inhibitor TOFA or the fatty acid synthase inhibitor cerulenin. Such FAS inhibition reduced the reduction potential of these cells, as indicated by increased NADH2+/NAD+ ratios under both in vitro and in vivo stroke conditions. As observed in the OGD model, FAS inhibition also resulted in increased cell death in the stroke model. Stroke mice treated with cerulenin did not only display increased brain injury but also showed reduced neurological recovery during the observation period of 4 weeks. Interestingly, cerulenin treatment enhanced endothelial cell leakage, reduced transcellular electrical resistance (TER) of the endothelium and contributed to poststroke blood-brain barrier (BBB) breakdown. The latter was a consequence of the activated NF-κB pathway, stimulating MMP-9 and ABCB1 transporter activity on the luminal side of the endothelium. In conclusion, FAS inhibition aggravated poststroke brain injury as consequence of BBB breakdown and NF-κB-dependent inflammation.
... Its efficacy was evaluated in a clinical trial (NCT02193334) in patients with acute SCI, with encouraging motor functional recovery (Nagoshi et al., 2020). Moreover, intrastriatal injections of HGF were successful in protecting neural progenitor cells from apoptosis in animal models of cerebral ischemia (Doeppner et al., 2011). Another way to deliver HGF is the use of hydrogel carriers, which are useful in supporting neurogenesis (Nakaguchi et al., 2012) and mitigate apoptosis and autophagy (Shang et al., 2010) after stroke in mice models. ...
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Hepatocyte growth factor (HGF) and its tyrosine kinase receptor, encoded by the MET cellular proto-oncogene, are expressed in the nervous system from pre-natal development to adult life, where they are involved in neuronal growth and survival. In this review, we highlight, beyond the neurotrophic action, novel roles of HGF-MET in synaptogenesis during post-natal brain development and the connection between deregulation of MET expression and developmental disorders such as autism spectrum disorder (ASD). On the pharmacology side, HGF-induced MET activation exerts beneficial neuroprotective effects also in adulthood, specifically in neurodegenerative disease, and in preclinical models of cerebral ischemia, spinal cord injuries, and neurological pathologies, such as Alzheimer’s disease (AD), amyotrophic lateral sclerosis (ALS), and multiple sclerosis (MS). HGF is a key factor preventing neuronal death and promoting survival through pro-angiogenic, anti-inflammatory, and immune-modulatory mechanisms. Recent evidence suggests that HGF acts on neural stem cells to enhance neuroregeneration. The possible therapeutic application of HGF and HGF mimetics for the treatment of neurological disorders is discussed.
Background Sufficient understanding of the systemic inflammatory response after stroke will make the therapeutic strategy targeting inflammation more feasible. Here, we aimed to identify the globally alterations of circulating cytokines in super-acute ischemic stroke (AIS). Methods A broad panel of 65 cytokines was measured in the plasma of twenty-eight AIS patients within 6 h after stroke onset (n = 28), cerebral hemorrhagic patients (n = 28) and healthy controls (n = 18). The diagnostic power of the candidate cytokines and their relationship with the number of lymphocytes and neutrophils were analyzed by receiver operating characteristic (ROC) and spearman rank correlation respectively. Results The expression level of plasma IL-1beta, IL-2, IL-2R, IL-5, IL-10, CD40L, HGF, MIP-3alpha and MMP-1 were obviously up-regulated, while IL-16 was down-regulated in AIS patients compared to healthy controls. Among them, IL-2R, IL-10, IL-16, MIP-3alpha, and MMP-1 were specially altered in AIS patients, while IL-1beta, IL-2, IL-5, CD40L and HGF were elevated simultaneously in AIS and hemorrhagic stroke patients. Interestingly, IL-6 and TNF-beta were found to be key facytors among the 65 cytokines to distinguish hemorrhage from ischemia. Furthermore, IL-1beta, IL-16, CD40L and HGF were obviously correlated with the number of lymphocytes, and IL-1beta and IL-16 were significantly associated with the number of neutrophils in AIS patients. These results suggest that lymphocytes and neutrophils associated inflammation may play a pivotal role in AIS. Conclusions Importantly, except for some mutual pathological processes, AIS and hemorrhage had their own distinctive pathogenesis, and transformation of this knowledge to further research may provide novel treatment strategy for AIS.
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Neurovascular remodeling has been recently recognized as a promising target for neurologic therapies. Hopes have emerged that, by stimulating vessel growth, it may be possible to stabilize brain perfusion, and at the same time promote neuronal survival, brain plasticity, and neurologic recovery. In this review, we outline the role of vascular endothelial growth factor (VEGF) in the ischemic brain, analyzing how this growth factor contributes to brain remodeling. Studies with therapeutic VEGF administration resulted in quite variable results depending on the route and time point of delivery. Local VEGF administration consistently enhanced neurologic recovery, whereas acute intravenous delivery exacerbated brain infarcts due to enhanced brain edema. Future studies should answer the following questions: (1) whether increased vessel density translates into improvements in blood flow in the hemodynamically compromised brain; (2) how VEGF influences brain plasticity and contributes to motor and nonmotor recovery; (3) what are the actions of VEGF not only in young animals with preserved vasculature, on which previous studies have been conducted, but also in aged animals and in animals with preexisting atherosclerosis; and (4) whether the effects of VEGF can be mimicked by pharmacological compounds or by cell-based therapies. Only on the basis of such information can more definite conclusions be made with regard to whether the translation of therapeutic angiogenesis into clinics is promising.
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Neural stem cells (NSCs) are present not only during the embryonic development but also in the adult brain of all mammalian species, including humans. Stem cell niche architecture in vivo enables adult NSCs to continuously generate functional neurons in specific brain regions throughout life. The adult neurogenesis process is subject to dynamic regulation by various physiological, pathological and pharmacological stimuli. Multipotent adult NSCs also appear to be intrinsically plastic, amenable to genetic programing during normal differentiation, and to epigenetic reprograming during de-differentiation into pluripotency. Increasing evidence suggests that adult NSCs significantly contribute to specialized neural functions under physiological and pathological conditions. Fully understanding the biology of adult NSCs will provide crucial insights into both the etiology and potential therapeutic interventions of major brain disorders. Here, we review recent progress on adult NSCs of the mammalian central nervous system, including topics on their identity, niche, function, plasticity, and emerging roles in cancer and regenerative medicine.
Hepatocyte growth factor (HGF), a natural ligand for the c-met protooncogene product, has mitogenic, motogenic and morphogenic activities for various cell types and functions as a organotrophic factor for regeneration of the liver, kidney and lung. We obtained evidence that HGF may function as a novel neurotrophic factor in the central nervous system. Northern blot analysis showed that 6 kb HGF mRNA and 9 kb c-Met/HGF receptor mRNA are expressed in various regions of the adult rat brain. In situ hybridization analysis revealed that intense hybridization signals for HGF mRNA were localized in cerebral cortex, hippocampus and amygdala. Consistently, specific localization of HGF protein in neurons of these regions was detected by immunohistochemical analysis and non-neuronal glial cells in cingulum, cerebellum, pons and medulla were also specifically stained. Specific intense hybridization signals for c-Met/HGF receptor mRNA were also widely distributed in the brain, including neurons of olfactory bulb, cerebral cortex, primary olfactory cortex, hippocampus and cerebellum. On the basis of the co-expression of HGF and c-Met/HGF receptor in hippocampal neurons, we found that HGF prolonged survival of embryonic hippocampal neurons in primary culture: HGF elicited maximal surviving effect at 0.5–1 ng/ml and the potency was comparable to that of nerve growth factor. More importantly, expression of both HGF and c-Met/HGF receptor mRNAs was markedly induced in response to cerebral ischemic injury. We propose that HGF functions as a neurotrophic factor in the central nervous system and that this neurotrophic function may have a role in the survival and reconstruction of specific neurons in response to cerebral injury.
Hepatocyte growth factor (HGF), a natural ligand for the c-met protooncogene product, exhibits mitogenic, motogenic, and morphogenic activities for regeneration of the liver, kidney, and lung. Recently, HGF was clearly shown to enhance neurite outgrowth in vitro. To determine whether HGF has a neuroprotective action against the death of neurons in vivo, we studied the effect of HGF on delayed neuronal death in the hippocampus after 5-minute transient forebrain ischemia in Mongolian gerbils. Continuous postischemic intrastriatal administration of human recombinant HGF (10 or 30 micrograms) for 7 days potently prevented the delayed death of hippocampal neurons under both anesthetized and awake conditions. Even when HGF infusion started 6 hours after ischemia (i.e., in a delayed manner), HGF exhibited a neuroprotective action. We conclude that HGF, a novel neurotrophic factor, has a profound neuroprotective effect against postischemic delayed neuronal death in the hippocampus, which may have implications for the development of new therapeutic strategies for ischemic neuronal damage in humans.
Neural precursor cells (NPC) are an interesting tool in experimental stroke research, but their therapeutic potential is limited due to poor long-term survival. We therefore in vitro transduced subventricular zone-(SVZ)-derived NPC with the anti-apoptotic fusion protein TAT-Bcl-x(L) and analyzed NPC survival, differentiation, and post-stroke functional deficits after experimental ischemia in mice. Survival of TAT-Bcl-x(L)-transduced NPC, which were injected at day 7 post-stroke into the ischemic striatum, was significantly increased at 4 weeks after stroke. Increased survival of NPC was associated with reduced infarct injury and decreased post-stroke functional deficits. Animals grafted with TAT-Bcl-x(L)-transduced NPC showed an increased number of immature cells expressing the neuronal marker doublecortin. Since mature neuronal differentiation of NPC was not observed, reduced post-stroke injury cannot be attributed to enhanced neuronal regeneration, but rather to indirect by-stander effects of grafted NPC. In line with this, NPC-mediated neuroprotection of cortical neurons in vitro was associated with increased secretion of growth factors. Thus, in vitro transduction of cultivated NPC with TAT-Bcl-x(L) results in enhanced resistance of transplanted NPC followed by long-term neuroprotection and ameliorated functional deficits after transient focal cerebral ischemia in mice.
Glial cell line-derived neurotrophic factor (GDNF) and hepatocyte growth factor (HGF) are strong neurotrophic factors, which function as antiapoptotic factors. However, the neuroprotective effect of GDNF and HGF in ameliorating ischemic brain injury via an antiautophagic effect has not been examined. Therefore, we investigated GDNF and HGF for changes of infarct size and antiapoptotic and antiautophagic effects after transient middle cerebral artery occlusion (tMCAO) in rats. For the estimation of ischemic brain injury, the infarct size was calculated at 24 hr after tMCAO by HE staining. Terminal deoxynucleotidyl transferase-mediated dUTP-biotin in situ nick end labeling (TUNEL) was performed for evaluating the antiapoptotic effect. Western blot analysis of microtubule-associated protein 1 light chain 3 (LC3) and immunofluorescence analysis of LC3 and phosphorylated mTOR/Ser(2448) (p-mTOR) were performed for evaluating the antiautophagic effect. GDNF and HGF significantly reduced infarct size after cerebral ischemia. The amounts of LC3-I plus LC3-II (relative to beta-tubulin) were significantly increased after tMCAO, and GDNF and HGF significantly decreased them. GDNF and HGF significantly increased p-mTOR-positive cells. GDNF and HGF significantly decreased the numbers of TUNEL-, LC3-, and LC3/TUNEL double-positive cells. LC3/TUNEL double-positive cells accounted for about 34.3% of LC3 plus TUNEL-positive cells. This study suggests that the protective effects of GDNF and HGF were greatly associated with not only the antiapoptotic but also the antiautophagic effects; maybe two types of cell death can occur in the same cell at the same time, and GDNF and HGF are capable of ameliorating these two pathways.
The importance of bioactive lipid signaling under physiological and pathophysiological conditions is progressively becoming recognized. The disparate distribution of sphingosine kinase (SphK) isoform activity in normal and ischemic brain, particularly the large excess of SphK2 in cerebral microvascular endothelial cells, suggests potentially unique cell- and region-specific signaling by its product sphingosine-1-phosphate. The present study sought to test the isoform-specific role of SphK as a trigger of hypoxic preconditioning (HPC)-induced ischemic tolerance. Temporal changes in microvascular SphK activity and expression were measured after HPC. The SphK inhibitor dimethylsphingosine or sphingosine analog FTY720 was administered to adult male Swiss-Webster ND4 mice before HPC. Two days later, mice underwent a 60-minute transient middle cerebral artery occlusion and at 24 hours of reperfusion, infarct volume, neurological deficit, and hemispheric edema were measured. HPC rapidly increased microvascular SphK2 protein expression (1.7+/-0.2-fold) and activity (2.5+/-0.6-fold), peaking at 2 hours, whereas SphK1 was unchanged. SphK inhibition during HPC abrogated reductions in infarct volume, neurological deficit, and ipsilateral edema in HPC-treated mice. FTY720 given 48 hours before stroke also promoted ischemic tolerance; when combined with HPC, even greater (and dimethylsphingosine-reversible) protection was noted. These findings indicate hypoxia-sensitive increases in SphK2 activity may serve as a proximal trigger that ultimately leads to sphingosine-1-phosphate-mediated alterations in gene expression that promote the ischemia-tolerant phenotype. Thus, components of this bioactive lipid signaling pathway may be suitable therapeutic targets for protecting the neurovascular unit in stroke.
Cerebral ischemia activates endogenous neurogenesis in the subventricular zone (SVZ) and the dentate gyrus. Consecutively, SVZ-derived neural precursors migrate towards ischemic lesions. However, functional relevance of activated neurogenesis is limited by poor survival of new-born precursors. We therefore employed the HI-virus-derived fusion protein TAT-Bcl-x(L) to study the effects of acute anti-apoptotic treatment on endogenous neurogenesis and functional outcome after transient cerebral ischemia in mice. TAT-Bcl-x(L) treatment led to significantly reduced acute ischemic cell death (128+/-23 vs. 305+/-65 TUNEL+ cells/mm(2) in controls) and infarct volumes resulting in less motor deficits and improved spatial learning. It significantly increased survival of doublecortin (Dcx)-positive neuronal precursors (389+/-96 vs. 213+/-97 Dcx+ cells in controls) but did not enhance overall post-ischemic cell proliferation or lesion-specific neuronal differentiation 28 days after ischemia. Our data demonstrate that post-stroke TAT-Bcl-x(L)-treatment results in acute neuroprotection, improved functional outcome, and enhanced survival of lesion-specific neuronal precursor cells after cerebral ischemia in mice.
Neural stem cells persist in the adult mammalian brain, within the subventricular zone (SVZ). The endogenous mechanisms underpinning SVZ neural stem cell proliferation, self-renewal, and differentiation are not fully elucidated. In the present report, we describe a growth-stimulatory activity of liver explant-conditioned media on SVZ cell cultures and identify hepatocyte growth factor (HGF) as a major player in this effect. HGF exhibited a mitogenic activity on SVZ cell cultures in a mitogen-activated protein kinase (MAPK) (ERK1/2)-dependent manner as U0126, a specific MAPK inhibitor, blocked it. Combining a functional neurosphere forming assay with immunostaining for c-Met, along with markers of SVZ cells subtypes, demonstrated that HGF promotes the expansion of neural stem-like cells that form neurospheres and self-renew. Immunostaining, HGF enzyme-linked immunosorbent assay and Madin-Darby canine kidney cell scattering assay indicated that SVZ cell cultures produce and release HGF. SVZ cell-conditioned media induced proliferation on SVZ cell cultures, which was blocked by HGF-neutralizing antibodies, hence implying that endogenously produced HGF accounts for a major part in SVZ mitogenic activity. Brain sections immunostaining revealed that HGF is produced by nestin-expressing cells and c-Met is expressed within the SVZ by immature cells. HGF intracerebroventricular injection promoted SVZ cell proliferation and increased the ability of these cells exposed in vivo to HGF to form neurospheres in vitro, whereas intracerebroventricular injection of HGF-neutralizing antibodies decreased SVZ cell proliferation. The present study unravels a major role, both in vitro and in vivo, for endogenous HGF in SVZ neural stem cell growth and self-renewal.