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Anitric oxide donor induces neurogenesis and reduces functional deficits after stroke in rats.

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A Nitric Oxide Donor Induces Neurogenesis
and Reduces Functional Deficits
After Stroke in Rats
Ruilan Zhang, MD,1 Li Zhang, MD,1 Zhenggang Zhang, MD, PhD,1 Ying Wang, MD,1 Mei Lu, PhD,1
Margot LaPointe, PhD,3 and Michael Chopp, PhD1,2
The adult rodent brain is capable of generating neuronal progenitor cells in the subventricular zone, and in the dentate
gyrus of the hippocampus, throughout the life of the animal. Signals that regulate progenitor cell proliferation, differentiation, and migration are not well known. We report that administration of a nitric oxide donor, (Z)-1-[N-(2aminoethyl)-N-(2-ammonioethyl) aminio]diazen-1-ium-1,2-diolate (DETA/NONOate), to young adult rats significantly
increases cell proliferation and migration in the subventricular zone and the dentate gyrus. Treatment with DETA/
NONOate also increases neurogenesis in the dentate gyrus. Furthermore, administration of DETA/NONOate to rats
subjected to embolic middle cerebral artery occlusion significantly increases cell proliferation and migration in the
subventricular zone and the dentate gyrus, and these rats exhibit significant improvements of neurological outcome
during recovery from ischemic stroke. Administration of DETA/NONOate significantly increases cortical levels of
guanosine monophosphate both in ischemic and nonischemic rats, supporting the role of nitric oxide in promoting cell
proliferation and neurogenesis. Thus, our data indicate that nitric oxide is involved in the regulation of progenitor cells
and neurogenesis in the adult brain. This suggests that nitric oxide delivered to the brain well after stroke may have
therapeutic benefits.
Ann Neurol 2001;50:602– 611
Functional recovery after stroke and brain injury may
be related to neurogenesis and compensatory responses
within the injured brain.1 How to induce neurogenesis
and promote functional recovery after neuronal injury
is an area of intense interest, with obvious clinical implications.1 Nitric oxide (NO) is a chemical messenger
in biological systems and serves as a neurotransmitter
in the brain. NO is synthesized from L-arginine in the
brain by three isotypes of NO synthase (NOS): neuronal (nNOS), endothelial, and inducible. During embryonic development, expression of nNOS increases in
the subventricular zone (SVZ) when it is formed at
E18, and migrating cells in and near the SVZ express
nNOS.2,3 In addition, elevation of NOS activity in the
developing cerebral cortical plate and hippocampus at
E15 to E19 correlates with the time course of cessation
of precursor cell proliferation and cell differentiation.2
These data suggest possible roles of NO in regulation
of the proliferation, differentiation, and migration of
progenitor cells in the developing brain.
We demonstrate that administration of an NO do-
nor to the rat significantly increases cell proliferation,
migration, and proliferation in the adult brain and significantly improves neurological outcome during recovery from ischemic stroke.
From the 1Department of Neurology, Henry Ford Health Sciences
Center, Detroit; 2Department of Physics, Oakland University,
Rochester; and 3Hypertension and Vascular Research Division,
Henry Ford Hospital, Detroit, MI.
Published online Sep 27, 2001; DOI: 10.1002/ana.1249
Received May 1, 2001, and in revised form Jul 17. Accepted for
publication Jul 18, 2001.
602
© 2001 Wiley-Liss, Inc.
Materials and Methods
All experimental procedures have been approved by the Care
of Experimental Animals Committee of Henry Ford Hospital.
Animal Model
Male Wistar rats weighing 320 to 380g were employed in
the present study. The middle cerebral artery (MCA) was
occluded by placement of an embolus at the origin of the
MCA.4
Experimental Protocol
To determine whether exogenous NO affects cell proliferation in nonischemic animals, under physiological conditions
we administered (Z)-1-[N-(2-aminoethyl)-N-(2-ammonioethyl) aminio]diazen-1-ium-1,2-diolate (DETA/NONOate),
Address correspondence to Dr Chopp, Department of Neurology,
Henry Ford Health Sciences Center, 2799 West Grand Boulevard,
Detroit, MI 48202. E-mail: chopp@neuro.hfh.edu
an NO donor with a half-life of 57 hours,5,6 to young adult
rats. Rats were randomly divided into four groups. Group 1
(single-Rx) rats received four consecutive intravenous (IV)
bolus doses of DETA/NONOate (0.1mg/kg each, every 15
minutes; total dose of 0.4mg/kg). Group 2 (two-Rx group)
rats received two treatments of DETA/NONOate (0.4mg/kg
for each dose), 24 hours apart. Group 3 (seven-Rx group)
rats received DETA/NONOate (0.4mg/kg) on the first experimental day, and DETA/NONOate (0.4mg/kg) was administered daily for an additional 6 consecutive days. Group
4 (control group) rats received four consecutive IV bolus of
saline.
To examine whether exogenous NO affects cell proliferation in ischemic animals, rats were subjected to embolic
MCA occlusion. Ischemic rats were randomly divided into
four groups. Group 1 (single-set precondition group) rats received four consecutive IV bolus of DETA/NONOate
(0.1mg/kg each, every 15 minutes; total dose of 0.4mg/kg) at
24 hours before embolization. Group 2 (two-set group) animals received four consecutive IV bolus doses of DETA/
NONOate (total dose of 0.4 mg/kg) 24 and 48 hours after
MCA occlusion. Group 3 (seven-set group) rats received four
consecutive bolus doses of DETA/NONOate (IV, 0.4mg/kg)
at 24 hours after embolization, and rats were injected intraperitoneally (IP) with DETA/NONOate (0.4mg/kg) daily
for an additional 6 consecutive days. Group 4 (control
group) rats were subjected MCA occlusion and received four
consecutive IV bolus of saline 24 hours after ischemia.
To examine whether administration of DETA/NONOate
affects nonischemic and ischemic brain cyclic guanosine
monophosphate (cGMP) levels, rats were randomly divided
into three groups. Group 1 (seven-Rx group) nonischemic
rats received four consecutive IV bolus doses of DETANONOate (0.1mg/kg each, every 15 minutes; total dose of
0.4mg/kg) on the first experimental day, and DETA/
NONOate (IP, 0.4mg/kg) was administered daily for an additional 6 consecutive days. Group 2 (seven-Rx group) rats
received four consecutive bolus doses of DETA/NONOate
(IV, 0.4mg/kg) 24 hours after embolization, and rats were
injected with DETA/NONOate (IP, 0.4mg/kg) daily for an
additional 6 consecutive days. Group 3 (control group) nonischemic rats received four consecutive IV bolus of saline. All
rats were sacrificed at 7 days after treatment. To examine
whether ischemia affects levels of cGMP, an additional group
of ischemic rats without any treatment was sacrificed from 1
to 42 days after ischemia, and levels of brain cGMP were
measured.
Immunohistochemistry
BrdU immunostaining was performed as previously described.8 To determine if BrdU immunoreactive cells express
neuronal phenotype, we performed double immunofluorescent staining for BrdU and neuronal marker NeuN, or microtubule associated protein-2 (MAP2) and glial fibrillary
acidic protein,8 a marker for astrocytes, on sections from animals at 42 days after treatment with DETA/NONOate.
Quantification
BrdU immunostained sections were digitized using a ⫻40
objective (Olympus BX40) via the microcomputer imaging
device computer imaging analysis system (Imaging Research,
St Catharines, Canada). BrdU immunoreactive nuclei were
counted on a computer monitor to improve visualization and
in one focal plane to avoid oversampling. Structures were
sampled either by selecting predetermined areas on each section (rostral migratory stream [RMS] and olfactory bulb
[OB] or by analyzing entire structures on each section (SVZ
and dentate gyrus). All BrdU immunoreactive-positive nuclei
in these areas are presented as the number of the BrdU immunoreactive cells per millimeter2 (data shown are mean ⫾
standard error). Density for the selected several sections was
averaged to obtain a mean density value for each animal.
Subventricular Zone
Every 40th coronal section was selected from each rat for a
total of seven sections between interaural ⫹ 10.6mm genu
corpus callosum, and interaural ⫹ 8.74mm anterior commissure crossing.9 BrdU immunoreactive-positive nuclei were
counted in the subventricular zone.
Rostral Migratory Stream and Olfactory Bulb
Every 20th section was selected from each rat for a total of 6
sections from the sagittal series of the OB/frontal cortex. As
depicted in Figure 1, two predetermined areas (100 ⫻
100␮m) in the RMS and four areas (300 ⫻ 300␮m) in the
granule cell layer (GCL) of the OB were analyzed on each
section.
Dentate Gyrus
Every 50th coronal section was selected from each rat for a
total of eight sections between interaural ⫹ 5.86mm and interaural ⫹ 2.96mm including the hilus, subgranular zone,
inner first to second and third of the GCL. The subgranular
zone, defined as a two-cell body wide zone along the border
of the GCL and the hilus, always was combined with the
GCL for quantification.
Bromodeoxyuridine Labeling
Bromodeoxyuridine (BrdU) was used as mitotic labeling to
measure frequency of cell proliferation.7 Animals received
daily IP injections of BrdU (50mg/kg, Sigma, St. Louis,
MO) on the first experimental day, and subsequently for 14
consecutive days. To determine if the proliferation and the
differentiation of cells in the SVZ and dentate gyrus of adult
rats is affected by NO, rats were sacrificed at 14 days (n ⫽
4 –5 per group) and 42 days (n ⫽ 4 –5 per group) after the
first dose of DETA/NONOate treatment, respectively.
Cyclic Guanosine Monophosphate Measurement in
Brain Tissue
We measured levels of cGMP in nonischemic and ischemic
rat brain. cGMP was determined by a commercially available
low pH immunoassay kit (R & D Systems, Inc., Minneapolis, MN). The sensitivity of the assay was approximately
0.6pmol/ml for the nonacetylated procedure. The brain was
rapidly removed and the cortex and the cerebellum were separated. The brain tissue was weighed and homogenized in 10
Zhang et al: NO Donor Effects After Stroke in Rats
603
volume of 0.1N HCl containing 1mM 3-isobutyl-1methylxanthine.
Behavioral Testing
An accelerating rotarod test and adhesive removal test were
employed to measure motor and somatosensory deficits.10,11
Behavioral tests were performed before MCA occlusion and
2, 4, 7, 14, 21, 28, 35, and 42 days after MCA occlusion by
an investigator blinded to the experimental groups.
The infarct volume was measured at 14 and 42 days after
ischemia as previously described.4
Statistical Analysis
The generalized estimation equations analysis approach was
used instead of the regular analysis of variance, because data
did not meet assumptions of normality and equal variance
for the analysis of variance. All values are presented as
means ⫾ standard error. Statistical significance was set at p
less than 0.05.
Results
Effects of Nitric Oxide on the Proliferation and
Migration of Cells in Nonischemic Adult
Subventricular Zone and Dentate Gyrus
Rats treated with DETA/NONOate had a significant
( p ⬍ 0.05) increase in numbers of BrdU immunoreactive cells in the SVZ compared with rats treated with
saline at 14 and 42 days after treatment (Fig 2A). Rats
treated with seven doses of DETA/NONOate exhibited the highest number of BrdU immunoreactive cells
among the three DETA/NONOate-treated groups at
14 days after treatment, which was significant ( p ⬍
0.01) compared with the number in the single-dose
group (see Fig 2A), suggesting that increases in BrdU
immunoreactive cells are dose-dependent.
Numbers of BrdU immunoreactive cells did not significantly increase in the RMS in rats treated with
DETA/NONOate at 14 and 42 days after treatment
(data not shown). However, significant increases in
BrdU immunoreactive cells were detected in the OB at
42 days after single DETA/NONOate treatment ( p ⬍
0.05), and at 14 and 42 days after two and seven sets
of DETA/NONOate treatment, compared with the
control group (see Fig 2B), suggesting an increased migration of SVZ progenitor cells.
Quantitative measurements of BrdU immunoreactive
cells in the dentate gyrus revealed that a single set of
DETA/NONOate treatment did not significantly increase numbers of BrdU immunoreactive cells at 14 and
42 days after treatment (see Fig 2C). In contrast, rats
treated with two and seven sets of DETA/NONOate exhibited significant increases in numbers of BrdU immunoreactive cells at 14 and 42 days after treatment ( p ⬍
0.01) compared with the control group (see Fig 2C).
These data demonstrate that the NO donor increases
cell proliferation in the dentate gyrus. The newborn progenitor cells in the dentate gyrus move from the sub-
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Fig 1. Sagittal view of the rat brain illustrating the anatomical sites of new cell proliferation in the subventricular zone
(SVZ), migration along the rostral migratory stream (RMS),
and differentiation in the olfactory bulb (OB). Two areas of
the RMS (small squares, 100 ⫻ 100␮m) and four areas
(300 ⫻ 300␮m) in the granule cell layer of the OB were
analyzed on each section. Color figure can be viewed online at
(www.interscience.wiley.com).
granular zone to the granular layers.12 We therefore analyzed the distribution of BrdU immunoreactive cells in
the different zones of the dentate gyrus. Treatment with
DETA/NONOate significantly decreased the percentage
of BrdU immunoreactive cells in the subgranular zone
( p ⬍ 0.05; see Fig 2D and E). However, the percentage
of BrdU immunoreactive cells in the granule layers significantly increased at 14 and 42 days after treatment
( p ⬍ 0.05) compared with the control group (see Fig
2D and E), indicating that NO promotes migration of
BrdU immunoreactive cells in the dentate gyrus.
The BrdU immunoreactive cells in the hilus were
triangular or irregular and smaller than nuclei of granular cells in the granule layers (Fig 3A), whereas the
BrdU immunoreactive cells were oval or round in the
granule layers and had the same size as nuclei of the
granule cells (see Fig 3B). BrdU immunoreactive cells
in the granular layers exhibited NeuN or MAP2 immunoreactivity (see Fig 3C–H). However, BrdU immunoreactive cells in the hilus showed glial fibrillary
acidic protein immunoreactivity, a marker for astrocytes (see Fig 3I–K).
Effects of Nitric Oxide on the Proliferation and
Migration of Cells in the Adult Ischemic Rat
Subventricular Zone and Dentate Gyrus
Embolic MCA occlusion resulted in significant ( p ⬍
0.05) increases in numbers of BrdU immunoreactive
Fig 2. Bar graphs show the proliferating cells in the SVZ (A), the OB (B), and the dentate gyrus in nonischemic rats treated with
different doses of DETA/NONOate (C). The percentage of distribution of BrdU immunoreactive cells in the dentate gyrus at (D)
14 and (E) 42 days after treatment with DETA/NONOate (*p ⬍ 0.05; **p ⬍ 0.01 vs the saline-treated group; and p ⬍ 0.01
vs the single-dose group).
cells in the ipsilateral SVZ and OB at 14 and 42 days
after MCA occlusion compared with nonischemic rats
but did not affect proliferation of cells in the contralateral SVZ and OB and in the both dentate gyrus, as we
previously demonstrated.8 However, significant ( p ⬍
0.05) increases in numbers of BrdU immunoreactive
cells were detected in both the contralateral and the
ipsilateral SVZ (Fig 4A and B), OB (see Fig 4C and
Zhang et al: NO Donor Effects After Stroke in Rats
605
Fig 3. BrdU immunoreactive cells were
(A) oval in the subgranular zone at 14
days and (B) round in the granule layers
42 days after treatment with DETA/
NONOate. Double immunofluorescent
staining shows that BrdU immunoreactive
cells (C, F, and I) were MAP2 (D),
NeuN (G), or glial fibrillary acidic protein immunoreactive (J). Merged image E
is from C and D; H from F and G; K
from I and J. Arrows indicate BrdU and
MAP2, BrdU and NeuN, or BrdU and
GFAP immunoreactive cells. Bar in B ⫽
10␮m for A and B; bar in H ⫽ 20␮m
for C–H; and bar in K ⫽ 50␮m for
I–K.
D), and dentate gyrus (see Fig 4E and F) at 14 and 42
days after MCA occlusion in rats treated with DETA/
NONOate compared with saline treated MCA occlusion group.
Effects of Nitric Oxide on Adult Brain Cyclic
Guanosine Monophosphate
Administration of DETA/NONOate for 7 days significantly ( p ⬍ 0.01) increased the cortical (Fig 5A) but
not the cerebellum (see Fig 5A) levels of cGMP compared with levels in the control group. Occlusion of
the MCA resulted in a significant increase in the ipsilateral cortical levels of cGMP 7 days after ischemia
(see Fig 5B). However, administration of DETA/
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NONOate to ischemic rats for 7 days significantly increased both the ipsilateral and the contralateral cortical levels of cGMP, compared with levels in ischemic
rats without treatment with DETA/NONO (see Fig
5C).
Effects of Nitric Oxide on Ischemic Lesion Volume;
Neurological Outcome
Rats in the DETA/NONOate and the saline-treated
groups showed marked motor impairment 2 days after
stroke. Partial recovery was detected in the DETA/
NONOate and saline-treated groups during 7 to 42
days of stroke (Fig 6A). However, the ischemic rats
treated with DETA/NONOate exhibited significant
Fig 4. Histograms show the effects of DETA/NONOate on proliferating cells in the SVZ (A and B), the OB (C and D), and the
dentate gyrus (E and F) in ischemic rats at 14 days (A, C, and E) and 42 days (B, D, and F) after ischemia (*p ⬍ 0.05 and
**p ⬍ 0.01 vs the saline-treated group).
improvements on the rotarod test at 4, 14, 28, 35, and
42 days after stroke ( p ⬍ 0.05) compared with the
saline-treated rats (see Fig 6A). Significant improvements on the adhesive removal test were detected in
the DETA/NONOate treated group at 2, 4, 7, 14, 28,
35, and 42 days after stroke (see Fig 6B). In addition,
animals in the DETA/NONOate-treated group exhibited a significant reduction of body weight loss at 35
and 42 days after stroke ( p ⬍ 0.05; see Fig 6C). However, there were no significant differences of infarct
volume between ischemic animals treated with DETA/
NONOate and animals in the control group at 14 and
42 days after ischemia (Fig 7).
Discussion
Our data demonstrate that administration of DETA/
NONOate to the young adult rat significantly increases proliferation of cells in the SVZ and dentate
gyrus, where neuronal progenitor cells are located.13–15
Proliferating cells exhibited a neuronal phenotype, suggesting that the NO donor enhances neurogenesis in
the young adult rat brain. Moreover, treatment of focal
Zhang et al: NO Donor Effects After Stroke in Rats
607
both in ischemic and nonischemic rats, indicating that
cGMP may contribute to the effects of NO on cell
proliferation and neurogenesis. To our knowledge,
these are the first data to demonstrate that NO contributes to tissue plasticity of brain, and this raises the
possibility that NO delivered to the brain well after
stroke may have therapeutic benefits.
The role of NO in the regulation of the proliferation
of cells in the adult brain has not been investigated.3 In
the developing rat cerebellum, NOS activity begins at
Fig 6. Line graph shows the effects of 7 doses of DETA/
NONOate treatment on the rotarod test (A), the adhesive
removal test (B), and body weight loss (C; *p ⬍ 0.05 and
**p ⬍ 0.01 vs the saline-treated group).
Fig 5. Histograms show cGMP levels in cerebral cortex and
cerebellum in nonischemic rats treated with DETA/NONOate
for 7 days (*p ⬍ 0.05 vs saline) (A), cGMP levels in the
cortex after MCA occlusion (*p ⬍ 0.05 vs nonischemic control rats) (B), and cGMP levels in the cortex in ischemic rats
with or without treatment with DETA/NONOate and sacrificed 7 days after ischemia (C; *p ⬍ 0.05 vs no ischemic control, #p ⬍ 0.05 vs MCAo).
cerebral ischemia in rats with DETA/NONOate enhanced proliferation of cells in the SVZ and in the
dentate gyrus, and significantly improved recovery of
neurological outcome. Administration of DETA/
NONOate significantly increased cortical levels cGMP
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Fig 7. Histogram shows ischemic lesion volume presented as a
percentage of lesion relative to the contralateral hemisphere in
the DETA/NONOate-treated and the saline-treated groups.
There is no statistical significance of infarct volume at 14
(hatched bar) and 42 (open bar) days after ischemia between
DETA/NONOate-treated and the saline-treated groups.
P3 and lasts as long as cellular proliferation occurs.2,16
Chronic administration of NG-nitro-l-arginine inhibits
NOS activity from P3 to P12, and decreases the number of granular cells in both the external and internal
granular layer of cerebellum at P12 in the rat.17 These
data indicate that NO may promote neuronal cell proliferation. However, studies on cultures of PC12 cells
show that NO inhibits cell proliferation when the differentiation process starts.18Using 14 days of BrdU labeling, we demonstrate that administration of DETA/
NONOate to young adult rats significantly increased
cell proliferation in the SVZ and the dentate gyrus.
Increases in proliferation of progenitor cells appear to
be induced by NO, because increases in the number of
BrdU immunoreactive cells are dose-dependent. In addition, we previously demonstrated that occlusion of
the MCA resulted in significant increases in proliferation of progenitor cells in the ipsilateral SVZ but not
in the dentate gyrus.8 In the present study, administration of DETA/NONOate to ischemic rats induced significant increases in numbers of BrdU immunoreactive
cells not only in SVZ but also in the dentate gyrus.
Furthermore, there are differences in the number of
BrdU cells between the ischemic and nonischemic rats
treated with DETA/NONOate. The ischemic rats
treated with DETA/NONOate had higher absolute
numbers of BrdU immunoreactive cells in the dentate
gyrus at 14 days and 42 days after MCA occlusion
than the nonischemic rats treated with DETA/
NONOate, suggesting that NO may amplify signals
generated by ischemia to increase proliferation of progenitor cells. Cells in the adult rodent SVZ and the
dentate gyrus are capable of proliferating throughout
the life of the animals.13–15 Progenitor cells express endothelial NOS and nNOS.19 NO can act as an amplifier of calcium signals in neuronal cells,20 and an increase of intracellular Ca2⫹ may promote cell
proliferation or protect neurons from developmental
cell death.21–23 Taken together, our data suggest that
NO may act as a pro-proliferative agent for progenitor
cells in the nonischemic and ischemic adult brains.
The progenitor cells in the SVZ of the lateral ventricle migrate into the OB and the newborn progenitor
cells in the dentate gyrus move from the subgranular
zone to the granular layers.12,24 Administration of
DETA/NONOate significantly increased numbers of
BrdU immunoreactive cells in the OB and in the granule layers at 42 days after treatment. During embryonic
development, expression of nNOS temporally coincides with neuronal migration in the rat cerebral cortex.25,26 Inhibition of NOS activity significantly decreases the migratory index of granule cells on
postnatal cerebellar slides.27 Our data suggest that NO
is involved in progenitor cell migratory process in the
adult brain.
NO is a potent activator of the soluble guanylate
cyclase and causes cGMP formation in the target
cells.28,29 In the rat hippocampus, the expression of
guanylyl cyclase is high in pyramidal neurons and dentate granule cells.30 That administration of DETA/
NONOate increases levels of cGMP indicates that the
drug increases NO in the brain. Our findings parallel
those of Estevez and colleagues, who found that
DETA/NONOate stimulates cGMP synthesis in neuron cultures.5 NO also regulates expression of transcriptional genes and some of these transcriptional
genes, such as the cyclic adenylic acid response
element-binding protein, are involved in cellular survival and neurogenesis.31,32
The present results show that functional improvement and increases in neurogenesis appear in parallel
after administration of DETA/NONOate, although
the ischemic lesion was not reduced. Whether the parallel appearance of functional improvements and neurogenesis is causally related is not yet known. However,
increases in neurogenesis in the dentate gyrus correlate
with improved learning for mice living in a rich environment.33–35 Functional recovery may relate to the
brain plasticity, and cGMP has been linked to changes
in axon extension and retraction; essential activities for
modifying patterns of neuronal connections.36
There have been numerous studies on the effects of
NO inhibitor and NO donors on stroke in a variety of
animal models.37,38 Nearly all these studies have addressed acute intervention after stroke.37,38 The neurotoxicity of NO in ischemic brain appears to result from
the simultaneous production of superoxide and NO
that leads to formation of peroxynitrite.38 NO gener-
Zhang et al: NO Donor Effects After Stroke in Rats
609
ated from nNOS or inducible NOS is neurotoxic,
whereas increases in endothelial NOS activity protect
against ischemic neuronal damage.38,39 Recent studies
both in vitro and in vivo indicate that NO produced
during neuronal ischemic preconditioning protects
against ischemic neuronal damage.31 Thus, NO can either be neuroprotective or neurotoxic depending on
the timing of exposure to NO.31,40
In summary, our data indicate that NO is involved
in regulation of progenitor cells and neurogenesis in
the adult brain and improves functional outcome after
stroke.
This work was supported by the National Institute of Neurological
Disorders and Stroke (PO1 NS23393 and RO1 NS35504).
We thank Cecylia Power and Gurocak Gulser for technical assistance.
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