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Proc Natl Acad Sci U S A. 2001 May 8; 98(10): 5874–5879. | PMCID: PMC33306 |
Copyright © 2001, The National Academy of Sciences Neurobiology FGF-2 regulation of neurogenesis in adult hippocampus after
brain injury Shinichi Yoshimura,*† Yasushi Takagi,*† Jun Harada,* Tetsuyuki Teramoto,* Sunu S. Thomas,* Christian Waeber,* Joanna C. Bakowska,‡ Xandra O. Breakefield,‡ and Michael A. Moskowitz*§ *Neuroscience Center, Department of Neurosurgery and Neurology, Massachusetts General Hospital, and ‡Molecular Neurogenetics Unit, Department of Neurology, Massachusetts General Hospital and Neuroscience Program, Harvard Medical School, Boston, MA 02129 Received January 22, 2001 Fibroblast growth factor-2 (FGF-2) promotes proliferation of
neuroprogenitor cells in culture and is up-regulated within brain after
injury. Using mice genetically deficient in FGF-2
(FGF-2−/− mice), we addressed the importance of
endogenously generated FGF-2 on neurogenesis within the
hippocampus, a structure involved in spatial, declarative, and
contextual memory, after seizures or ischemic injury. BrdUrd
incorporation was used to mark dividing neuroprogenitor cells and NeuN
expression to monitor their differentiation into neurons. In the
wild-type strain, hippocampal FGF-2 increased after either kainic acid
injection or middle cerebral artery occlusion, and the numbers of
BrdUrd/NeuN-positive cells significantly increased on days 9 and 16
as compared with the controls. In FGF-2−/− mice, BrdUrd
labeling was attenuated after kainic acid or middle cerebral artery
occlusion, as was the number of neural cells colabeled with both BrdUrd
and NeuN. After FGF-2−/− mice were injected
intraventricularly with a herpes simplex virus-1 amplicon vector
carrying FGF-2 gene, the number of BrdUrd-labeled cells increased
significantly to values equivalent to wild-type littermates after
kainate seizures. These results indicate that endogenously
synthesized FGF-2 is necessary and sufficient to stimulate
proliferation and differentiation of neuroprogenitor cells in the adult
hippocampus after brain insult. Keywords: fibroblast growth factor, cerebral
ischemia, seizure, gene delivery The dogma that neuronal
replacement is not possible after brain injury has been challenged by
recent data showing that mammalian neuroprogenitor cells in the adult
brain can proliferate and differentiate into neurons ( 1– 7). For
example, neuroprogenitor cells within the hippocampus divide and
differentiate into neurons after cerebral ischemia or seizures ( 8– 11).
Little is known about what initiates and promotes this potentially
therapeutic response in vivo, but growth factors and
neurotrophins have been implicated in culture studies ( 12– 18).
Fibroblast growth factor-2 (FGF-2) and epidermal growth factor
stimulate mitogenesis of cultured neuroprogenitor cells, whereas
neurotrophin-3 and brain-derived neurotrophic factor enhance neuronal
differentiation ( 12– 14). When maintained in medium containing FGF-2,
hippocampal progenitor cells from the adult rat brain proliferate and
differentiate into neurons and glia in culture ( 19). FGF-2 also is
up-regulated within the hippocampus after brain injuries, for example,
after focal cerebral ischemia ( 20, 21) or after seizure induced by
kainic acid ( 22– 25). However, the functional role of FGF-2 in
neurogenesis has not been explored before or after brain injury
in vivo. Understanding the factors that control neurogenesis
in vivo is critical to therapeutic manipulation of this
phenomenon for application to neurodegenerative disorders. Here, we examined the impact of endogenously generated
FGF-2 on neurogenesis in the dentate gyrus of the hippocampus after
kainate-induced seizures and cerebral ischemia by using mice
genetically deficient in FGF-2. We compared the extent of
neuroprogenitor cell proliferation by using BrdUrd incorporation into
replicating DNA, and differentiation of newly born cells into neurons
and glia by using immunocytochemical markers in these knockout animals
with and without vector-mediated delivery of FGF-2. We now show that
BrdUrd incorporation is diminished, as compared with wild type, in mice
deficient in FGF-2 after kainate-induced seizures or cerebral ischemia,
and that vector-mediated delivery of FGF-2 to the hippocampus
stimulates BrdUrd incorporation and subsequent differentiation of
neuroprogenitor cells into neurons to near wild-type levels. Animals. FGF-2 knockout mutant mice (FGF-2 −/− mice) and
their wild-type littermates (FGF-2 +/+ mice) were
generated from two heterozygous mating pairs
(FGF-2 +/−, SV129/Black Swiss background)
(generously provided by Thomas Doetschman, University of Cincinnati
College of Medicine, Cincinnati, ref. 26). Mice were genotyped by PCR
using primers specific for the wild-type and the FGF-2 knockout
alleles. Male FGF-2 −/− mice and
FGF-2 +/+ mice were used at 8–10 weeks of age. Animal care and experimental protocols complied with The Principles of
Laboratory Animal Care (Guide for the Care and Use of Laboratory
Animals, National Institutes of Health). Before all operations or
injections, mice were anesthetized with 3% isoflurane and maintained
on 1.5% isoflurane in 70% N2O and 30%
O2 by using a Fluotec 3 vaporizer (Colonial
Medical, Amherst, NH). In kainate experiments, control animals were
injected with vehicle (saline); in middle cerebral artery occlusion
(MCAO) experiments, sham controls were anesthetized with exposure of
the carotid artery only. After these procedures, the mice were kept at
37°C for 2 h until awake. Kainic Acid Injection. Intraperitoneal kainic acid injection (20 mg/kg; dissolved at 10
mg/ml in sterile PBS, Sigma) caused overt seizures 5–10 min
later, which lasted for ≈2 h. Seizures were scored as follows: 1,
arrest of motion; 2, myoclonic jerks of the head and neck, with brief
twitching movements; 3, unilateral clonic activity; 4, bilateral
forelimb tonic and clonic activity; and 5, generalized tonic-clonic
activity with loss of postural tone including death from continuous
convulsions, as described ( 27). One death occurred among 30 animals for
each of FGF-2 +/+ and
FGF-2 −/− groups. MCAO. Cerebral ischemia was performed in spontaneously ventilating mice, as
described ( 28, 29). Body temperature was maintained at ≈37°C with a
thermostat (FHC, Brunswick, ME). Regional cerebral blood flow was
measured by laser-Doppler flowmetry (PF2B; Perimed, Stockholm) ( 28,
29). The left MCA was occluded with an 8–0 nylon monofilament
(Ethicon, Somerville, NJ) coated with a mixture of silicone resin
(Xantopren, Bayer Dental, Osaka) and a hardener (Elastomer Activator,
Bayer Dental), as described ( 28, 29). Twenty minutes later, the
filament was withdrawn, and reperfusion was confirmed by laser-Doppler
flowmetry. Infarct area was quantified on sections stained by an image-analysis
system (M4, Imaging Research, St. Catherines, Ontario, Canada) and
calculated by summing the volumes of each section ( 28, 29). BrdUrd Injections. Animals received i.p. injections of BrdUrd (50 mg/kg; dissolved
at 5 mg/ml in sterile PBS, Sigma). Two daily injections were
given on days 6 and 7 followed by a single injection on day 8 after
brain injury, kainate, or MCAO. The animals were killed 1 or 28 days
after the last BrdUrd injection (i.e., days 9 or 35). In other animals,
BrdUrd was given twice a day on days 13 and 14 followed by a single
injection on day 15, and then killed on day 16. For histological
evaluation, the animals were perfused transcardially with 4%
paraformaldehyde in phosphate buffer under deep anesthesia. Physiology. In randomly selected animals ( n = 4–5 for each group),
mean arterial blood pressure were monitored as described ( 28, 29).
Arterial blood samples were analyzed for oxygen
(PaO 2) and carbon dioxide
(PaCO 2) before and during ischemia by using a
blood gas/pH analyzer (Corning 178, Ciba Corning Diagnostics,
Medford, MA). Preparation of Herpes Simplex Virus-1 (HSV-1) Amplicon Vector. Mouse FGF-2 cDNA in the plasmid pBluescript (ATCC no. 63348) was
released by digestion with NotI/ApaI and inserted
into the NotI/ApaI site of pSecTag2/Hygro B
(Invitrogen), so as to add an N-terminal secretion signal from the
V-J2-V region of the mouse Ig kappa-chain. Next, mouse FGF-2 cDNA with
the secretion signal was digested out from the pSecTag2/Hygro B
construct with NheI/ XhoI and ligated
between the NheI/ XhoI sites in the
multicloning site of the HSV-1 amplicon, pHGCX (kindly provided by
Yoshinaga Saeki, Massachusetts General Hospital, ref. 30). This
amplicon, pHGCX/mFGF2 contains both an enhanced green
fluorescent protein cassette driven by the HSV IE 4/5 promoter
and an FGF-2 cassette driven by the immediate early human
cytomegalovirus promoter. Helper virus-free amplicon vector stocks of
HSV-1/no FGF2 (empty) and HSV-1/mFGF-2 were prepared as
described ( 31, 32). Briefly, amplicon DNA was cotransfected with DNA
from the cosmid set C6Δa48Δa into Vero2–2 cells by using the
Lipofectamine protocol (GIBCO/BRL). Crude supernatant was
harvested, and virions were purified by sucrose gradient,
centrifugation. Viral titers were determined by infecting Vero2–2
cells that are transduced by these amplicons with high efficiency and
by monitoring numbers of green fluorescent protein-positive cells
24 h later. The vector stocks contained 10 7
to 10 8 transducing units/ml. Virus Injection. Virus injection was performed 1 day before kainic acid injection in
anesthetized mice placed in a stereotaxic apparatus (Stoelting, Wood
Dale, IL). A 1-mm burr hole was made at 1.4 mm lateral and 0.6 mm
caudal to bregma. A 26-gauge needle (10-μl Hamilton syringe) was then
stereotactically inserted into the right ventricle at a depth of 1.7 mm
from the dura. Ten microliters of vector (5 ×
10 7 transducing units/ml) were injected
over 20 min (0.5 μl/min) with a stereotaxic injector
(Stoelting). The needle was left in place for 5 min and then slowly
withdrawn to minimize cerebrospinal fluid leakage ( 33). Immunohistochemistry. Immunohistochemistry was performed on free-floating 50-μm coronal
sections pretreated by denaturing DNA, as reported ( 1– 4, 16). We used
mouse anti-BrdUrd (Becton Dickinson), 1:400, or rat anti-BrdUrd ascites
fluid (Harlan Sera-Lab, Loughborough, U.K.) for double labeling, 1:100;
rabbit glial fibrillary acidic protein antibody-cy3 conjugated (Sigma),
1:2,500; and mouse anti-NeuN (Chemicon), 1:200. To determine the number
of BrdUrd-labeled cells, we stained for BrdUrd by using the peroxidase
method (ABC system, with biotinylated horse anti-mouse IgG
antibodies and diamino-benzidine as chromogen; Vector Laboratories).
The fluorescent secondary antibodies used were FITC-labeled anti-rat
IgG and cy3-labeled anti-mouse IgG (Jackson ImmunoResearch), 1:200. Stereology. BrdUrd-positive cells were counted in the dentate gyrus in four
sections per animal (one of every 12th serial, 50 μm sections) by
using a ×40 objective throughout the rostro caudal extent of the
granule cell layer. The granule cell layer area
(mm2) was measured on adjacent sections stained
with cresyl violet. The total granule cell volume
(mm3) was estimated by summing the traced
granule cell areas for each section multiplied by the distance between
sampled sections. The number of BrdUrd-labeled cells per dentate gyrus
then was calculated from the sectional and total volumes of the granule
cell layer. Enzyme Immunoassay (EIA). For EIA, the brains were removed without transcardial perfusion and the
hippocampi were dissected and frozen immediately at −80°C. Stored
hippocampi were homogenized and centrifuged at 14,000 ×
g for 30 min at 4°C. Protein concentration of each
supernatant was determined by a protein assay kit (Bio-Rad). EIA for
FGF-2 was performed by using an assay kit (Quantikine HS, R&D Systems)
according to the manufacturer's instruction. Statistical Analysis. Values are expressed as the mean ± SD. ANOVA with Bonferroni's
posthoc analysis in STATVIEW 5.0 for Macintosh was
used for statistical analysis throughout the study. P values
< 0.05 were considered statistically significant. To measure the extent of neurogenesis in the dentate gyrus after
injury, the number of cells showing BrdUrd incorporation into the
nuclei of dentate granule cells was assessed. When administered i.p. to
naive (control) mice, sparse labeling was observed (Figs.
and 2).
The numbers of BrdUrd-positive cells in naive
FGF-2 +/+ and FGF-2 −/−
mice did not differ (943 ± 388 and 858 ± 157 in
FGF-2 +/+ and FGF-2 −/−
mice, respectively). Levels of FGF-2 were below a detection limit (5
pg/mg protein) in FGF-2 −/− mice, whereas
levels of around 85 pg/mg protein were found in
FGF-2 +/+ hippocampus (Table
1). Kainic acid administration enhanced
BrdUrd labeling in both FGF-2 +/+ and
FGF-2 −/− mice, although on day 9 the increase
was much less in the FGF-2 −/− mice
(FGF-2 +/+: 11-fold,
FGF-2 −/−: 3.4-fold); and on day 16 an increase
in labeling was observed only in FGF-2 +/+
littermates (Fig. ). After kainic acid injection, mice were evaluated
for seizure activity according to the previously described scoring
system. Accordingly, seizure scores for FGF −/−
mice did not differ significantly from FGF-2 +/+
littermates (2.1 + 0.8 and 2.0 + 1.4 at 15 min; 3.6 + 1.1 and 4.0
+ 1.4 at 45 min; 2.4 + 1.0 and 2.3 + 0.8 at 90 min in
FGF-2 +/+ and FGF-2 −/−,
respectively) as assessed at the specified time points. Kainic acid
significantly raised the levels of hippocampal FGF-2 from baseline to
279 ± 96 pg/mg protein in FGF-2 +/+
strain at 1 day after ( P < 0.01). This finding
suggests that FGF-2 is important for proliferation of progenitor cells
in the dentate gyrus after kainic acid administration.
| Table 1FGF-2 concentration in hippocampus after viral
gene transfer |
To determine whether the BrdUrd labeling in
FGF-2−/− mice was stimulus specific, BrdUrd
incorporation into dentate gyrus cells also was examined after
reversible (20 min) unilateral MCAO in FGF-2+/+
littermates and FGF-2 mutants. Again, the increase in labeling was less
in FGF-2−/− mice (2.0-fold) than
FGF-2+/+ mice (3.8-fold) on day 9, and this
difference persisted on day 16. Hippocampal FGF-2 levels were increased
from 107 ± 26 to 258 ± 98 pg/mg protein 7 days after
MCAO (P < 0.01) in FGF-2+/+
mice, with again no change in measured levels in knockout mice. There
were no significant differences in physiological findings before
operation (96 ± 11 and 94 ± 11 mmHg for mean arterial blood
pressure; 7.30 ± 0.1 and 7.29 ± 0.01 for pH; 33 ± 7
and 35 ± 6 for PaC02, in
FGF-2+/+ and FGF-2−/−
animals, respectively) or 10 min after MCAO (94 ± 10 and 80
± 11 mmHg for mean arterial blood pressure; 7.32 ± 0.08 and
7.29 ± 0.05 for pH; 34 ± 9 and 39 ± 10 for
PaCO2 in FGF-2+/+ and
FGF-2−/−, respectively). Regional cerebral
blood flow measured by laser-Doppler flowmetry during and after MCAO
did not differ between groups [9.1 ± 3.7% and 8.4 ± 2.2
during ischemia in FGF-2+/+ and
FGF-2−/−, respectively and 96.3 ± 24.8%
and 88.0 ± 29.7% after ischemia in
FGF-2+/+ and FGF-2−/−,
respectively] (n = 5 per group). Further, there was no
significant difference in the volume of cerebral infarction, located
mainly in lateral striatum (FGF-2+/+: 7.8 ±
2.3%; FGF-2−/−: 7.6 ± 2.2% of
ipsilateral hemisphere, n = 6 per group), which
therefore did not contribute to differences in number of BrdUrd-stained
cells between FGF-2+/+ and
FGF-2−/− mice. We next investigated the fate of BrdUrd-positive cells to determine
whether the lack of FGF-2 influenced the extent of differentiation of
labeled cells in the dentate gyrus at a later time point (35 days after
injury). No differences in the number of BrdUrd-positive cells were
detected between strains under basal conditions. After kainate
administration or ischemic injury, the number of BrdUrd-labeled cells
was higher (6.8-fold in FGF-2 +/+ and 2.4-fold in
FGF-2 −/− mice after kainate; 4.2-fold in
FGF-2 +/+ and 1.6-fold in
FGF-2 −/− mice after MCAO), as detected at early
time points (Table 2). After kainate
administration or MCAO, a significantly greater proportion of
BrdUrd-positive cells were colabeled with NeuN in
FGF-2 +/+ mice, as compared with sham controls,
and knockouts (Table 2). Glial fibrillary acidic protein staining
infrequently colocalized with BrdUrd-positive cells within the granular
zone (Fig. ).
| Table 2Number and phenotype of
BrdUrd-positivecells |
To determine whether endogenous FGF-2 expression was
critical for enhancing neurogenesis in FGF-2 −/−
mice, we used FGF-2 gene delivery with an HSV-1 amplicon vector in both
FGF-2 +/+ mice and
FGF-2 −/− mice. As compared with mice injected
with HSV-1/empty vector, those injected with HSV-1/mFGF-2 vector
showed increased FGF-2 concentration in the hippocampus (Table 1).
BrdUrd-positive cells were increased about 2-fold in both
FGF-2 +/+ mice (1,002 ± 254 and 2282 ±
234 cells/dentate gyrus, HSV-1/empty and
HSV-1/mFGF-2, respectively), and
FGF-2 −/− (1,016 ± 300 and 2,109 ±
764 cells/dentate gyrus, HSV-1/empty and
HSV-1/mFGF-2, respectively) (Fig.
). After FGF-2 gene transfer and kainate
challenge, the number of BrdUrd-positive cells on day 9 increased about
30% in FGF-2 +/+ mice (HSV-1/empty,
9,389 ± 617; HSV-1/mFGF-2, 12,147 ± 1,155
cells/dentate gyrus), and about 5-fold in the knockout strain
(HSV-1/empty, 2,462 ± 590; HSV-1/mFGF-2, 9,866
± 1,636 cells/dentate gyrus). To examine the fate of the
BrdUrd-positive cells after FGF-2 gene transfer, we used
FGF-2 +/+ mice killed on day 35, which had been
injected with BrdUrd on days 6, 7, and 8 after injury. The number of
BrdUrd-positive cells in FGF-2 +/+ mice under
resting conditions was increased 30% by the FGF-2 gene delivered by
HSV-1 vector (HSV-1/empty, 436 ± 56; HSV-1/mFGF-2,
592 ± 107 cells/dentate gyrus, P < 0.05)
and about 40% after kainate seizures (HSV-1/empty, 3,117.8
± 619; HSV-1/mFGF-2, 4,395 ± 598 cells/dentate
gyrus, P < 0.01). These data indicate that on-site
FGF-2 expression can enhance both insult-induced and basal BrdUrd
incorporation and neuronal differentiation in dentate gyrus cells of
FGF-2 −/− and FGF-2 +/+
mice.
This study confirms the pivotal role of FGF-2 in
proliferation and differentiation of the progenitor cells in the adult
hippocampus in response to injury. In response to kainic acid-induced
seizures and cerebral ischemia, FGF-2−/− mice
showed low levels of neurogenesis, measured as BrdUrd-positive cell
number, as compared with FGF-2+/+ mice. This
decrease in neurogenesis in mutant mice could be overcome by gene
delivery of FGF-2, which stimulated proliferation of neuroprogenitor
cells in the dentate gyrus, and also augmented proliferation in
FGF-2+/+ mice after insult. These results suggest
that endogenously generated FGF-2 is necessary and
sufficient to trigger a cascade of neurogenesis-related events in
dentate gyrus after brain insult. Interestingly, under basal condition, there was no significant
difference in the number of BrdUrd-positive cells in the dentate gyrus
between FGF-2+/+ and
FGF-2−/− mice, despite apparent differences in
FGF-2 concentration in the hippocampus. Hence, under resting
conditions, factors other than FGF-2 must maintain low levels of
proliferation of neuroprogenitor cells in dentate gyrus. Importantly, gene transfer of FGF-2 and an associated secretion signal
increased the number of BrdUrd-positive cells in kainate-treated
FGF-2 −/− animals as compared with untreated
animals. Normally FGF-2 does not have a signal sequence for cell
secretion through the Golgi apparatus ( 34– 37), and it is probably
released extracellularly only after cell damage. According to this
hypothesis, it is speculated that FGF-2 plays a negligible role in the
normal state, but with increasing damage, more FGF-2 is released and
stimulates neurogenesis. This explanation may account for the reason we
observed differences in the number of BrdUrd-positive cells between
strains only after ischemia and kainate seizures, and why the FGF
vector increased neurogenesis after injury. In other studies,
recombinant FGF-2 protein administered intraventricularly or s.c. did
not stimulate dentate gyrus neurogenesis in the adult rat brain ( 16,
38). There are two possible explanations for these apparent
differences: ( i) In the present study, expression of FGF-2
with secretion signal sequence was driven by a strong viral promoter
via an HSV-1 amplicon vector. Therefore, this system confers robust and
constant FGF expression to a number of cell types not only at the
injection site, but also to other cells via retrograde transport ( 31,
39, 40). Therefore the levels and distribution of FGF-2 are different
from that achieved by protein injection. ( ii) Other factors
that are activated by FGF-2 gene transfer, such as activin A, might
alter the effect of FGF-2 on progenitor cell proliferation. Activin A,
a member of transforming growth factor-β superfamily, regulates the
neuroprotective action of FGF-2 in vivo ( 41). In our
preliminary experiments, activin A was increased in the hippocampus
after kainate administration. Therefore, gene delivery of FGF may
stimulate BrdUrd incorporation by augmenting expression of other
intracellular growth factors. Regarding FGF-2 release from cells, plasminogen activator-mediated
proteolysis provides a mechanism for the dissociation of biologically
active FGF-2-heparan sulfate complexes from the extracellular matrix
( 34– 37). Recently, cerebral ischemia and kainate seizures were shown
to activate plasminogen activator ( 42– 46). We therefore speculate that
brain injury may not only up-regulate synthesis of FGF-2
intracellularly, but promote cell secretion and dissociation from
extracellular matrix. Moreover, ischemia and kainate seizures both
up-regulate expression of an FGF-2 receptor in brain ( 21, 22, 24, 25).
Therefore, regulation and transportation of FGF-2 may critically
regulate neurogenesis after brain injury. In this study, we used kainate injection and focal cerebral ischemia as
neuronal injury models. In ischemia, increased activation of excitatory
amino acid receptors, including the
α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid/kainate
receptor, is considered to be a major cause of neuronal damage
( 47– 49). Both types of injury up-regulate neurogenesis, with a greater
increase in the BrdUrd-positive cells in the hippocampus after kainic
acid treatment as compared with cerebral ischemia. In fact, inhibition
of excitatory amino acid receptors can decrease cell proliferation in
the dentate gyrus during ischemia ( 50). On the other hand, cell death
may be one of the triggering factors for neurogenesis ( 51). We found no
significant difference between FGF-2 +/+ and
FGF-2 −/− mice in infarction volume 7 days after
MCAO, suggesting that the extent of cell death external to hippocampus
did not account for differences in BrdUrd-positive cell numbers. DNA
fragmentation (terminal deoxynucleotidyltransferase-mediated dUTP nick
end labeling) and Fluorojade-B staining for degenerating cells were
similar in both strains on days 1, 2, and 7 after kainate and MCAO.
Furthermore, cell death in the dentate gyrus may not be an essential
stimulus to promote neurogenesis in the dentate gyrus, because animals
receiving a low dose of kainic acid (10 mg/kg) did exhibit
milder seizures and an increase in BrdUrd-positive cell number, but did
not show any detectable cell death in the hippocampus (data not shown). Increased amount and/or availability of FGF-2 appears to
be critical to neurogenesis after brain injury. In normal animals, the
total concentration of FGF-2 increased and peaked at 1 day after kainic
acid treatment to 2.5-fold normal levels and returned to the basal
level 7 days later, whereas the level of FGF-2 after MCAO was higher
(2.4-fold sham-operated levels) on day 7 than on day 3. Rapid increase
in the FGF-2 levels after kainic acid treatment may correspond to the
robust increase in BrdUrd-positive cell number, with MCAO having less
of an increased number on day 9. When levels of FGF-2 were increased by
vector-mediated delivery, neurogenesis was stimulated for both strains
under control and injury paradigms, with the greater stimulus to
neurogenesis in knockout mice after the kainate insult. Previous studies have shown that FGF-2 −/− mice
do not exhibit gross developmental defects in brain morphology ( 26),
although they do show a reduced density of neurons in sensory-motor
cerebral cortex, but not in the striatum or cerebellum ( 52, 53). In the
present study, we found no significant differences in volume of the
granule cell layer (FGF-2 +/+; 3.24 ± 0.17,
FGF-2 −/−; 3.39 ± 0.29, n
= 6 each) or density of granule cells in dentate gyrus
(FGF-2 +/+; 1.82 ± 0.17,
FGF-2 −/−; 1.79 ± 0.21, n
= 6 each) between wild-type and knockout animals. These results suggest
that other factors regulate or can compensate for lack of FGF-2 in the
normal development of the dentate gyrus. In conclusion, we have shown that overexpression of FGF-2 increases
neurogenesis, whereas FGF-2 deficiency decreases neurogenesis in the
adult brain in response to injury. Hence, FGF-2 is a critical regulator
of neuronal repair. Insights into the importance of FGF-2 after brain
injury provide a strategy for understanding mechanisms of repair or
regeneration within the central nervous system after exposure to
toxins, stroke, seizures, or during neuroodegenerative disease.
Supplementation of FGF-2 in the brain after injury should help promote
neurogenesis, and this can be achieved by gene delivery. This work was supported by National Institutes of Health
Interdepartmental Stroke Program Project 5 P50 NS10828 (M.A.M.),
National Institute of Neurological Disorders and Stroke Grant NS24279
(X.O.B.), and National Institute of Mental Health Grant MH60587
(X.O.B.). S.Y. and Y.T. were supported by Japan Society for the
Promotion of Science fellowships. | | FGF-2 | fibroblast growth factor-2 | | | MCAO | middle cerebral
artery occlusion | | | HSV-1 | herpes simplex virus-1 |
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