Download Induced Spreading Depression Evokes Cell Division of

Induced Spreading Depression Evokes Cell Division of
Astrocytes in the Subpial Zone, Generating Neural
Precursor-Like Cells and New Immature Neurons in the
Adult Cerebral Cortex
Jing-Hui Xue, MD, PhD; Hiroji Yanamoto, MD, DMSc; Yukako Nakajo, MSc;
Norimitsu Tohnai, PhD; Yoshikazu Nakano, BSc; Takuya Hori, MCE;
Koji Iihara, MD, PhD; Susumu Miyamoto, MD, PhD
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Background and Purpose—New immature neurons appear out of the germinative zone, in cortical Layers V to VI, after
induced spreading depression in the adult rat brain. Because neural progenitors have been isolated in the cortex, we set
out to determine whether a subgroup of mature cells in the adult cortex has the potential to divide and generate neural
precursors.
Methods—We examined the expression of endogenous markers of mitotic activity, proliferating cell nuclear antigen, and
vimentin as a marker for neuronal progenitor cells, if any, in the adult rat cortex after spreading depression stimulation.
Immunohistochemical analysis was also performed using antibodies for proliferating cell nuclear antigen, for vimentin,
and for nestin. Nestin is a marker for activity dividing neural precursors.
Results—At the end of spreading depression (Day 0), glial fibrillary acidic protein-positive cells in the subpial zone and
cortical Layer I demonstrated increased mitotic activity, expressing vimentin and nestin. On Day 1, nestin⫹ cells were
found spreading in deeper cortical layers. On Day 3, vimentin⫺/nestin⫹, neural precursor-like cells appeared in cortical
Layers V to VI. On Day 6, new immature neurons appeared in cortical Layers V to VI. Induced spreading depression
evokes cell division of astrocytes residing in the subpial zone, generating neural precursor-like cells.
Conclusions—Although neural precursor-like cells found in cortical Layers V to VI might have been transferred from the
germinative zone rather than the cortical subpial zone, astrocytic cells in the subpial zone may be potent neural
progenitors that can help to reconstruct impaired central nervous system tissue. Special caution is required when observing
or treating spreading depression waves accompanying pathological conditions in the brain. (Stroke. 2009;40:e606-e613.)
Key Words: astrocytes 䡲 ischemia 䡲 neural stem cells 䡲 neurophysiology 䡲 neuroregeneration 䡲 stroke management
䡲 stroke recovery
S
more, induced SD generates new immature neurons, at an
ectopic location, in cortical Layers V to VI.6 Recently, it was
found that induced SD activates neurogenesis in the hippocampal subgranular zone.7 In the embryonic ventricular zone, spontaneously occurring propagations of “calcium waves” accelerate
cell division of radial glial cells (neural progenitors).8 “Calcium
waves” initiated at the sperm entry site in mammalian eggs have
been related to levels of cell cycle regulating factors.9 These
evidences suggest that SD, SD-like depolarizing waves, or
calcium waves are an endogenous activation signal for germinative cells and neural progenitors to divide.
tatus epilepticus and cerebral ischemia stimulate persistent neurogenesis by increasing divided cell density in the
germinative zone and promoting a majority of them to
neuronal differentiation.1,2 Spreading depression (SD), consisting of transient increase in [Ca⫹⫹]i, is triggered by
epileptic seizures, cerebral injury, or ischemic stroke.3 SD has
been also associated with migraine with aura.4,5
Previously, we found that induced multiple SD waves in the
adult rat brain under normal condition activates cell division of
neural progenitor cells in the subventricular zone (SVZ), enhancing persistent neurogenesis toward the olfactory bulb.6 Further-
Received June 12, 2009; final revision received July 31, 2009; accepted August 4, 2009.
From the Lab for Cerebrovascular Disorders (J.-H.X., H.Y., Y. Nakajo, N.T., Y. Nakano, T.H.), Research Institute of National Cardio-Vascular Center
(NCVC), Suita, Osaka, Japan; the Department of Cerebrovascular Surgery (H.Y., K.I., S.M.), NCVC, Suita, Osaka, Japan; the Research Laboratory (Y.
Nakano), Rakuwakai Otowa Hospital, Kyoto, Japan; the Department of Neurosurgery (J.-H.X.), First Affiliated Hospital, General Hospitals of PLA,
Beijing, PR China; Hakuju (T.H.), Institute for Health Science, Tokyo, Japan; the Department of Cardiovascular Science (H.Y., Y. Nakajo), Osaka
University Graduate School of Medicine, Suita, Osaka, Japan; and the Department of Materials and Life Science (N.T.), Osaka University Graduate
School of Engineering, Suita, Osaka, Japan.
Correspondence to Hiroji Yanamoto, MD, DMSc, National Cardiovascular Center, 5-7-1 Fujishirodai, Suita, Japan 565-8565. E-mail
[email protected]
© 2009 American Heart Association, Inc.
Stroke is available at http://stroke.ahajournals.org
DOI: 10.1161/STROKEAHA.109.560334
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Figure 1. A, The KCl infusion site, at Bregma ⫺1.0 mm, for the induction of SD. B, The region of interest, at Bregma ⫹0.2 mm, used
for immunohistochemical analysis. Ndl indicates needle (site); SSp, primary somatosensory area; SSs, supplemental somatosensory
area; FPC, frontoparietal cortex; CPu, caudate putamen; V–VI, cortical Layers V to VI; cc, corpus callosum; LV, lateral ventricle; ACg,
anterior cingulate cortex. C, The monitoring of direct current potential in the cortex during KCl infusion detected repetitive SD waves.
In the adult mammalian brain, outside of the germinative
zone, neural progenitors have been isolated from the septum,
striatum, subcortical white matter, and cortex.10 –12 We hypothesized that neural progenitors may reside permanently in
the cortex and investigated whether a subgroup of cells in the
adult cortex undergoes mitosis after induced SD expressing
the marker for neural progenitors. The absence of mitotic
figure and neural progenitor-like cells in the cortex after
induced SD confirms that new immature neurons migrate
from outside the cortex. We analyzed mRNA levels for
proliferating cell nuclear antigen (PCNA), an endogenous
marker for mitotic activity,13 and vimentin,14 a cytoskeletal
intermediate filament protein primarily localized in radial
glial cells in the embryonic brain.
Nestin belongs to another class of cytoskeletal intermediate
filament protein recognized as a general marker of proliferating neural precursor cells.15 Neural precursor cells express
nestin abundantly as do radial glia in the embryonic central
nervous system,16 but most of these disappear after completion of the development of the central nervous system. In the
adult SVZ, actively dividing cells generating clusters of
neuroblasts are neural precursors, an intermediate proliferating population between glial fibrillary acidic protein-positive
neural progenitors and neuroblasts.6,17 We also studied the
temporospatial distribution of cells expressing PCNA, vimentin, nestin, and doublecortin (DCX) in the adult rat cortex
after induced SD.
Methods
The experimental protocols were approved by the Experimental
Animal Research committee at the National Cardio-Vascular Center.
Every effort was made to minimize the suffering and number of
animals used. In all, 116 male Sprague-Dawley rats (SLC, Kyoto,
Japan), 8 to 9 weeks old, were used. All rats were allowed free access
to food and water before and after all procedures.
Induction of SD
SD was generated by a continuous microinfusion of 4 mol/L KCl at
a rate of 1.0 ␮L/hour for 48 hours into the left cortex (Figure 1).
Cortical direct current potential was monitored starting 3 hours after
the pump implantation (n⫽5) using an Ag/AgCl electroencephalogram needle electrode.18,19
Northern Blot Analysis
Setting Day 0 as the day of KCl pump removal, groups of 6 rats were
euthanized (under deep anesthesia) on Days 1, 3, 6, and 12 after
48-hour SD as were saline infusion control rats on Days 1, 3, 6, and
12 after a 48-hour vehicle infusion (n⫽4) and untreated normal
controls that received no infusion (n⫽6). Total RNA was separated
from the left cortex using the standard agarose gel electrophoresis
technique. A cDNA probe for PCNA20 and vimentin21 was prepared
by reverse transcription–polymerase chain reaction. The hybridized
signals ([32P]-dCTP) were semiquantified (Image Gauge; Fujiphoto
Film). Signals were compared among the groups. A DNA probe
complementary to 28S rRNA was used to confirm equal loading (5
␮g) of the RNA samples.
Immunohistochemistry
On Days 0, 1, 3, 6, and 12 after 48-hour KCl infusion (n⫽6 each),
after 48-hour vehicle infusion (n⫽4 each), and without the pump
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Results
Induction of SD
Multiple SD waves, 7⫾1 episodes/hour, appeared on the
direct current potential monitor (Figure 1).
Northern Blot Analysis
The probe detected the PCNA or vimentin mRNA in a single
band (Figure 2). The baseline signals in normal cortex
increased by 2.2-fold or 2.1-fold on Day 1. By contrast, these
signals did not increase throughout the observation period in
the vehicle group (Figure 2).
Figure 2. Expression levels of PCNA and vimentin mRNA. Signals for PCNA and vimentin mRNA increased in the cortex after
induced SD, but not after vehicle infusion. N indicates untreated
normal control.
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implantation (n⫽4), brains were removed, cut into 7 2-mm-thick
coronal slabs, and immersed in methanol Carnoy’s solution. The
paraffin-embedded slab rostral to the KCl injection site was sliced
into 7 sets of 6 3-␮m-thick coronal sections 21 ␮m apart (Figure 1B).
In addition, 3 sets of 6 3-␮m-thick coronal sections, 21 ␮m apart,
were also obtained using a separate set of animals on Days 1, 3, 6,
and 12 after a 48-hour KCl infusion (n⫽4 each). The slice thinness
precludes any pseudopositive double-labeled images.
Expression of PCNA, nestin, vimentin, glial fibrillary acidic
protein (GFAP), and neuronal nuclei was visualized using monoclonal
antibodies against PCNA (1:80; mouse-PC10; Upstate Biotechnology),
nestin (1:100; mouse-R-401; Becton, Dickinson and Company), vimentin (1:150; mouse-V9; Sigma), GFAP (1:100; mouse-GF-2; Dako),
and neuronal nuclei (1:200; mouse-antineuronal nuclei; Chemicon).
The immunostaining techniques in details were described elsewhere.6 Hematoxylin was used as a counterstain.
To identify the characteristics of dividing cells, we performed
double immunostaining with alkaline phosphatase-labeled streptavidin for the visualization for GFAP following the primary
diaminobenzidine-labeling for PCNA. In the analysis of the PCNA⫹
cell density, we tallied only astrocytic large-sized clear nuclei, but
not microglia-like small-sized dense nuclei ⬍5 ␮m (Figure 3A,
bottom right) in this study.
To identify apoptotic cells, and migrating neuroblasts, brain slices
were stained using terminal deoxynucleotidyl transferase-mediated
dUTP nick end labeling (TUNEL; ApopTag; Serologicals Co), or
DCX (1:200; guinea pig-anti-DCX; Chemicon), respectively, at
Days 1, 3, 6, and 12 after a 48-hour KCl infusion.
5-Bromo-2ⴕ-deoxyuridine Administration
All rats undergoing immunohistochemical study received the S-phase
marker, 5-bromo-2⬘-deoxyuridine (BrdU; B-5002; Sigma) through
drinking water (1 mg/mL) starting on the day of pump implantation.6
Double immunostaining was performed using monoclonal mouse
anti-BrdU (1:150; 3D4; BD Biosciences) for divided cells and
polyclonal rabbit anticlass III ␤-tubulin (␤-tubulinIII; 1:200;
PRB-435P; BabCO) for cells committed to a neuronal lineage.6
Quantification and Statistical Analysis
The numbers of PCNA⫹ cells, cells in mitosis with visualized chromosomes, vimentin⫹, nestin⫹ cells, and BrdU⫹/␤-tubulinIII⫹ cells were
counted at 21-␮m intervals, 6 sections per animal at each time point, at
600⫻ magnification using image analysis software (Mac Scope; Mitani
Co, Osaka, Japan). The targeted region, within a 1200⫻600-␮m square,
was set in cortical Layers I to VI (Figure 1B). The area within a 50-␮m
distance from the pia mater was defined as the subpial zone (SPZ). Cell
densities/mm3 were calculated after stereological corrections.6 Statistical analysis was performed using one-way analysis of variance. If
multiple comparisons were indicated, the Bonferroni t test was applied.
Data are presented as mean⫾SEM.
Immunohistochemistry
The laminar structures visualized by neuronal nuclei stain
were not affected by induced SD (Figure 3A).6 On Day 0,
PCNA⫹ cells were found primarily in the SPZ or cortical
Layer I and to a lesser extent in cortical Layers II to VI, and
cells in mitosis were observed in the SPZ (Figures 3B, 4A,
and 5A). On Day 1, PCNA⫹ cells or dividing cells were
found spreading in cortical Layers I to VI or in cortical Layers
I to IV, respectively. On day 3, the total number of these cells
decreased significantly (Figure 4A–B). There was no expression of PCNA or mitotic figure in the cortex of untreated
normals (Figure 3B), in the contralateral hemisphere after
induced SD, or rats treated with vehicle infusion (data not
shown). PCNA⫹ cells and mitotic figure were identified as
GFAP⫹ and Neu/U⫺ (Figures 3E and 5B–E).
In normal cortex, immunoreactivity toward vimentin was
detected only in the arachnoid tissues and pia mater (Figure
3C). On Day 0, cells in the SPZ and cortical Layer I expressed
vimentin (Figures 3C and 6A–B). On Day 1, the vimentin⫹
cells decreased in the SPZ and increased in the deeper cortical
Layer I. Within the SPZ, the vimentin⫹ cell density decreased
from Day 0 to 3 and increased from Day 3 to 6 (Figure 4C).
Vimentin⫹ cells underwent mitosis (Figure 6C–D).
Immunoreactivity for nestin was not observed in normal or
vehicle-treated cortex (data not shown). On Day 0, cells in the
SPZ and cortical Layer I expressed nestin (Figures 3D and 6E).
On Day 1, the predominant distribution of nestin⫹ cells shifted
from the SPZ to cortical Layer I, and nestin⫹ cells were
observed in cortical Layers II to VI (Figures 3D and 4D). Mitotic
figures were observed in nestin⫹ cells (Figures 3D and 6F–H).
In cortical Layers V to VI on Day 3, the nestin⫹ cell density was
higher than the vimentin⫹ cell density (P⬍0.001; Figure 4C–D),
indicating that nestin⫹/vimentin⫺ cells appeared there. Nestin⫹
cells disappeared on Day 6, but the appearance of vimentin⫹
cells lasted at least for 12 days (Figure 4C–D).
Immunoreactivity for DCX was detected in migrating
neuroblasts in the SVZ or rostral migratory stream, but not in
the cortex after induced SD during the observation period
(Figure 3G). TUNEL-positive cells were not observed in the
cortical layers during the observation period (Figure 3H).
BrdU⫹/␤-tubulinIII⫹ cells were found in cortical Layers V
to VI after induced SD (Figures 4E and 5F, G, K), showing
significant increase on Days 6 and 12 compared with untreated normal or vehicle controls. The spatiotemporal distribution of new immature neurons is illustrated in Figure 3F.
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Figure 3. Histological findings after induced SD. A, Neuronal nuclei⫹ cells. Mature neurons and cortical architecture visualized in the
normal cortex (N) and in the cortex after induced SD. B, The appearance of PCNA⫹ cells. On Day 0 (d 0) after induced SD, PCNA⫹
cells appeared primarily in cortical Layer I, spreading to deeper cortical layers on Day 1. C, The appearance of vimentin⫹ cells. On Day
0, vimentin⫹ cells appeared in cortical Layer I extending over cortical Layers I to IV on Day 3. D, The appearance of nestin⫹ cells. Cells
residing in the SPZ and a subgroup of cells in the adjacent cortical Layer I expressed nestin on Day 0, extending to cortical Layers V to
VI on Day 3. A–D, magnification: ⫻100, left panels; ⫻600, right panels, originally. Bar: 100 ␮m left panels; 20 ␮m, right panels. E,
GFAP⫹ or vimentin⫹ cells. In normal cortex, cells in the SPZ (N, arrows) and cortical Layer I express GFAP (upper 4 panels). GFAP⫹
mitotic figures in the SPZ on Day 0 (d0, arrows). A GFAP⫹ cell in cortical Layer I on Day 3 (d3, arrow) extending its cellular process
toward the pia matter. GFAP⫹ cells underlining the pia matter on Day 6 (d6, arrows). A cellular process extending toward deeper cortical layers (large arrow). Vimentin⫹ cells underlining the pia matter on Day 6 (bottom panel, arrows). A vimentin⫹ cellular process
extending from the pia matter (large arrow) on Day 6. F, The distribution of new immature neurons. BrdU⫹/␤-tubulinIII⫹ cells (dots)
observed only in cortical Layers V to VI in the region of interest (indicated by the square in the left cortex, see Figure 1) after induced
SD. Each diagram is an integrated view of 6 animals; a superimposed composite of 6 brain sections (one section from one animal) on
Day 3, 6, or 12. BrdU⫹/␤-tubulinIII⫹ cells were not detected in the untreated normal or the vehicle group. E–F: magnification: 600⫻,
originally. Bar: 20 ␮m. G, DCX⫹ cells and (H) analysis with TUNEL stain. DCX⫹ (brown) cells were not detected in cortical Layers I to VI
for 12 days after induced SD. Migrating neuroblasts in the rostral migratory stream (left) or SVZ (right; on Day 6) served as a positive
control (magnified window). TUNEL⫹ (brown) cells were not detected in the cortex for 12 days after induced SD (counterstain: methyl
green). TUNEL⫹ apoptotic cell bodies in blood clot around the needle cavity (on Day 3) served as a positive control (magnified window). G–H: magnification: ⫻100, originally. Bar: 100 ␮m, ⫻600 in the magnified window. Bar: 20 ␮m.
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Figure 4. The density of PCNA⫹ cells, mitotic figures, vimentin⫹, or nestin⫹ cells in the cortical layers after induced SD. A, From Day 0
to 1, the PCNA⫹ cell density decreased in the SPZ (P⬍0.001) and increased in the adjacent cortical Layer I (P⫽0.002) or in cortical
Layers V to VI (P⫽0.018), but not in cortical Layers II to IV (P⫽0.061). The density in all layers decreased from Day 1 to 3 (P⬍0.001). B,
From Day 0 to 1, the distribution of mitotic figures shifted from the SPZ toward the adjacent cortical Layer I or Layers II to IV. C, From
Day 0 to 1, the vimentin⫹ cell density decreased in the SPZ (P⬍0.001) but increased in the adjacent cortical Layer I (P⫽0.008). From
Day 1 to 3, the density in cortical Layers II to IV increased (P⫽0.001), but decreased from Day 3 to 6 (P⬍0.001). From Day 3 to 6, the
density in the SPZ increased (P⫽0.003). D, From Day 0 to 1, the nestin⫹ cells density in the SPZ decreased (P⬍0.001) but increased in
the adjacent cortical Layer I (P⬍0.001). The density in all layers increased from Day 0 to 3 (P⫽0.002), an effect that disappeared at Day
6. E, BrdU⫹/␤-tubulinIII⫹ cells increased in cortical Layers V to VI on Days 6 and 12 compared with untreated normal animals or vehicle
control (P⬍0.05). N indicates untreated normal control; d1, 3, 6, and d12, 1, 3, 6, and 12 days after SD. Asterisk: P⬍0.05; double
asterisk: P⬍0.01; triple asterisk: P⬍0.001; significantly different from untreated controls.
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Figure 6. Characteristics of vimentin⫹ or nestin⫹ cells. A vimentin⫹
cell (arrow) extending a cellular process to the pia matter (on the
right) on Day 0 (A) and Day 1 (B). A vimentin⫹ mitotic cell at
metaphase in cortical Layers II to IV on Day 1 (C). A postmitotic
(asymmetrical division-like) cell with doubled nuclei with and
without vimentin-like immunoreactivity in the perikarya in cortical
Layers V to VI at Day 1 (D). Asymmetrical cell division has been
defined as a describable difference in daughter cells.35 A nestin⫹
cell in cortical Layer I extending its cellular process toward the
pia matter on Day 0 (E, arrow). A nestin⫹ mitotic cell at metaphase (F) or cytokinesis (G) in cortical Layers II to IV on Day 1.
A nestin⫹ postmitosis-like cell in cortical Layers V to VI on Day
3 (H). Magnification: 600⫻, originally. Bar: 10 ␮m.
Figure 5. Phenotype of PCNA⫹ or mitotic cells and early committed or mature neurons. A, A PCNA⫹ (brown) cell (on the left) is
extending a cellular process toward the pia mater (on the right) on
Day 0 after induced SD. B, PCNA⫹ (brown) cells express GFAP
(red) in cortical Layers II to IV on Day 1. C–D, GFAP⫹ (red) cells
with visualized chromosomes in cortical Layers II to IV. E, Cells in
mitosis were negative for neuronal nuclei. Representative images
of new immature neurons (F–G, arrow) expressing ␤-tubulin (red)
and BrdU (brown) in cortical Layers V to VI. Native mature neurons
(H–I, arrow) expressing ␤-tubulin (red) but not BrdU (brown). Representative confocal laser scan images of native mature neurons (J,
arrows) expressing ␤-tubulin (red) but not BrdU (green). A new
immature neuron (K) expressing ␤-tubulin (red) and BrdU (green).
Magnification 600⫻, originally. Bar: 10 ␮m.
Discussion
Induced SD stimulated GFAP⫹ astrocytes residing in cortical
Layer I to express vimentin (the marker for radial glia or
neural progenitor cells) and/or nestin (the marker for neural
progenitor or precursor cells) and divide. Cell division in
vimentin⫹ and/or nestin⫹ cells from Day 0 to 1 was considered to be the cause of increased vimentin⫹ or nestin⫹ cell
density in all layers on Day 3. The appearance of vimentin⫹
cells for at least 12 days after the enhanced gene expression
on Day 1 demonstrated a relatively long life for vimentin.
From Day 0 to 3, the vimentin⫹ cell density in the SPZ
decreased but increased in the other cortical layers. Because
there was no sign of active apoptosis in the SPZ from Day 1
to 3, the acute decrease in vimentin⫹ cell density was not
considered to be caused by apoptotic cell loss in the SPZ but
the transfer of these cells to other cortical areas.
Actively dividing neural progenitors in the embryonic
ventricular zone have the characteristics of radial glia.22
Radial glial cells and GFAP⫹ neural progenitors in the SVZ
express vimentin.17,23 Radial glial cells divide asymmetrically, producing neural precursor cell(s) and a new radial glial
cell.24 The radial process of radial glia serves as a scaffold
connected to the embryonic ventricular surface and the pia
mater in embryonic cortical Layer I to guide their daughter
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cells to migrate into cortical Layers II to VI.24 The long
processes of radial glia cells remain attached to these surfaces
even during mitosis.25 When the neuronal production is
complete, after “final asymmetrical” cell division with loss of
vimentin, most of the radial glial cells transform into mature
astrocytes, and only a few radial glial cells remain into
adulthood as relatively quiescent neural progenitors in the
SVZ.17,24
The vimentin⫹ cell density increased from Day 3 to 6
without a prior increase in mitotic cell density in the SPZ
(Figure 4A), suggesting that vimentin⫹ cells spreading over
cortical Layers II to IV moved to the SPZ. To clarify
translocation mechanisms, if any, including scaffold structures involving the pia mater, requires further investigation.
After induced SD, DCX, the marker for migrating neuroblasts, was not detected in the cortex or the corpus callosum
after induced SD, indicating that new immature neurons
(newborn neuroblasts) do not migrate from the germinative
zone through corpus callosum to the cortex. It has also been
reported that newly generated immature neurons found in the
normal adult rat neocortex do not express DCX, indicating
that new neurons are derived from neural precursors in the
cortex under normal conditions.26 Taken together, these
observations make it appear likely that ␤-tubulinIII⫹/BrdU⫹
cells (divided and early committed neurons, ie, new immature
neurons) appearing in the adult cortex were generated from
nestin⫹/vimentin⫺ (neural precursor) cells appearing at the
same location.
It is interesting that vimentin⫹ radial glia-like cells appeared in cortical Layer I, the oldest structure in the cortex,
under which new neurons appear in the embryonic period
generating cortical Layer VI to II in an inside-out gradient.27
Besides the SPZ in the cortex, actively dividing cells in
“cerebellar” SPZ continue to generate neuroblasts beyond
puberty in rabbits.28 In the developing dentate gyrus, the SPZ
in the hippocampus has been identified as a transient neurogenic zone to form subgranular zone.29 Thus, the SPZ in the
cerebellum and the hippocampus carry germinative cells, at
least temporarily, in the developing brain. A germinative cell
population, if resident in the SPZ in the adult cerebral cortex
and even surviving in the vicinity of cortical lesion development after stroke or cerebral injury, may help to reconstruct
injured cortex by providing new neurons.
Compared with the present results, SD waves were less
frequent (1.7⫾0.5 /h) when direct current potential was
monitored a day after KCl pump implantation in our
previous study.18 In line with this, the frequency of SD
waves decreased 6 hours after a continuous KCl injection,
from 12 per hour to 2 per hour.30 Therefore, we estimate
the total number of SD waves as 84⫾26/48 hours in the
present study.
SD waves are frequently observed in the cortex of patients
with ischemic stroke, subarachnoid hemorrhage, or traumatic
cerebral injury.31,32 SD or SD-like waves triggered by experimental focal ischemia, 52 to 78 acute events in 24 hours,
have recruited penumbral tissue into the infarct core.33 In
contrast, KCl-induced multiple SD waves can induce ischemic tolerance through brain-derived neurotrophic factor, a
gene-dependent mechanism.19 Increase in BDNF levels and
appearance of immature new neurons induced by multiple SD
waves may play a beneficial role to the brain function. Thus,
special caution is required when inducing, observing or
treating SD waves in the brain.
Recently, GFAP-␦, an isoform of GFAP, was found in
astrocytes residing in the SVZ, the subgranular zone in the
hippocampus, and the SPZ in the adult human cortex.34
GFAP-␦, unlike GFAP-␣, plays no role in astrogliosis, but
has great benefits in migration.34 This indicates that astrocytes in the SPZ are a different cell population from the other
astrocytes in the brain.
Although it is still uncertain from the present data
whether nestin⫹/vimentin⫺ cells appearing in cortical
Layers V to VI are neural precursors originating in cortical
Layer I, we propose that the astrocytes residing in the SPZ
in the adult cortex are latent or quiescent neural progenitors. We also propose that multiple SD waves are an
endogenous activation signal for germinative cells in the
brain to divide.
Acknowledgments
We are thankful for the valuable assistance of Koji Nishimori,
Nozomi Momosaki, Tomoko Furuta, and Eri Shiozuka. We are also
thankful for the valuable suggestions from Keiji Machino and
Takemasa Bando.
Sources of Funding
The study was supported by grants from MEXT of Japan, and the
Japan Health Sciences Foundation.
Disclosures
None.
References
1. Parent JM, Yu TW, Leibowitz RT, Geschwind DH, Sloviter RS,
Lowenstein DH. Dentate granule cell neurogenesis is increased by
seizures and contributes to aberrant network reorganization in the adult
rat hippocampus. J Neurosci. 1997;17:3727–3738.
2. Liu J, Solway K, Messing RO, Sharp FR. Increased neurogenesis in the
dentate gyrus after transient global ischemia in gerbils. J Neurosci.
1998;18:7768 –7778.
3. Somjen GG. Mechanisms of spreading depression and hypoxic spreading
depression-like depolarization. Physiol Rev. 2001;81:1065–1096.
4. Pearce JMS. Is migraine explained by Leao’s spreading depression?
Lancet. 1985;2:763–766.
5. Bigal ME, Kurth T, Hu H, Santanello N, Lipton RB. Migraine and
cardiovascular disease: possible mechanisms of interaction. Neurology.
2009;72:1864 –1871.
6. Yanamoto H, Miyamoto S, Tohnai N, Nagata I, Xue JH, Nakano Y,
Nakajo Y, Kikuchi H. Induced spreading depression activates persistent
neurogenesis in the subventricular zone, generating cells with markers for
divided and early committed neurons in the caudate putamen and cortex.
Stroke. 2005;36:1544 –1550.
7. Urbach A, Redecker C, Witte OW. Induction of neurogenesis in the adult
dentate gyrus by cortical spreading depression. Stroke. 2008;39:
3064 –3072.
8. Weissman TA, Riquelme PA, Ivic L, Flint AC, Kriegstein AR. Calcium
waves propagate through radial glial cells and modulate proliferation in
the developing neocortex. Neuron. 2004;43:647– 661.
9. Sardet C, Roegiers F, Dumollard R, Rouviere C, McDougall A. Calcium
waves and oscillations in eggs. Biophys Chem. 1998;72:131–140.
10. Palmer TD, Ray J, Gage FH. FGF-2-responsive neuronal progenitors
reside in proliferative and quiescent regions of the adult rodent brain. Mol
Cell Neurosci. 1995;6:474 – 486.
11. Palmer TD, Markakis EA, Willhoite AR, Safar F, Gage FH. Fibroblast
growth factor-2 activates a latent neurogenic program in neural stem
Xue et al
12.
13.
14.
15.
16.
17.
18.
Downloaded from http://stroke.ahajournals.org/ by guest on June 18, 2017
19.
20.
21.
22.
Neural Progenitor-Like Cells in the Adult Subpial Zone
cells from diverse regions of the adult CNS. J Neurosci. 1999;19:
8487– 8497.
Nunes MC, Roy NS, Keyoung HM, Goodman RR, McKhann GII, Jiang
L, Kang J, Nedergaard M, Goldman SA. Identification and isolation of
multipotential neural progenitor cells from the subcortical white matter of
the adult human brain. Nat Med. 2003;9:439 – 447.
Matsumoto K, Moriuchi T, Koji T, Nakane PK. Molecular cloning of
cDNA coding for rat proliferating cell nuclear antigen (PCNA)/cyclin.
EMBO J. 1987;6:637– 642.
Alonso G. Proliferation of progenitor cells in the adult rat brain correlates
with the presence of vimentin-expressing astrocytes. Glia. 2001;34:
253–266.
Lendahl U, Zimmerman LB, McKay RD. CNS stem cells express a new
class of intermediate filament protein. Cell. 1990;60:585–595.
Frederiksen K, McKay RD. Proliferation and differentiation of rat neuroepithelial precursor cells in vivo. J Neurosci. 1988;8:1144 –1151.
Morshead CM, Reynolds BA, Craig CG, McBurney MW, Staines WA,
Morassutti D, Weiss S, van der Kooy D. Neural stem cells in the adult
mammalian forebrain: a relatively quiescent subpopulation of subependymal cells. Neuron. 1994;13:1071–1082.
Yanamoto H, Hashimoto N, Nagata I, Kikuchi H. Infarct tolerance
against temporary focal ischemia following spreading depression in rat
brain. Brain Res. 1998;784:239 –249.
Yanamoto H, Xue JH, Miyamoto S, Nagata I, Nakano Y, Murao K,
Kikuchi H. Spreading depression induces long-lasting brain protection
against infarcted lesion development via BDNF gene-dependent
mechanism. Brain Res. 2004;1019:178 –188.
Ino H, Chiba T. Expression of proliferating cell nuclear antigen (PCNA)
in the adult and developing mouse nervous system. Brain Res Mol Brain
Res. 2000;78:163–174.
Kindy MS, Bhat AN, Bhat NR. Transient ischemia stimulates glial
fibrillary acid protein and vimentin gene expression in the gerbil neocortex, striatum and hippocampus. Brain Res Mol Brain Res. 1992;13:
199 –206.
Noctor SC, Flint AC, Weissman TA, Wong WS, Clinton BK, Kriegstein
AR. Dividing precursor cells of the embryonic cortical ventricular zone
have morphological and molecular characteristics of radial glia.
J Neurosci. 2002;22:3161–3173.
e613
23. Dahl D, Rueger DC, Bignami A, Weber K, Osborn M. Vimentin, the
57 000 molecular weight protein of fibroblast filaments, is the major
cytoskeletal component in immature glia. Eur J Cell Biol. 1981;24:
191–196.
24. Malatesta P, Appolloni I, Calzolari F. Radial glia and neural stem cells.
Cell Tissue Res. 2008;331:165–178.
25. Corbin JG, Gaiano N, Juliano SL, Poluch S, Stancik E, Haydar TF.
Regulation of neural progenitor cell development in the nervous system.
J Neurochem. 2008;106:2272–2287.
26. Dayer AG, Cleaver KM, Abouantoun T, Cameron HA. New GABAergic
interneurons in the adult neocortex and striatum are generated from
different precursors. J Cell Biol. 2005;168:415– 427.
27. Marin-Padilla M. Dual origin of the mammalian neocortex and evolution
of the cortical plate. Anat Embryol (Berl). 1978;152:109 –126.
28. Ponti G, Peretto P, Bonfanti L. A subpial, transitory germinal zone forms
chains of neuronal precursors in the rabbit cerebellum. Dev Biol. 2006;
294:168 –180.
29. Li G, Kataoka H, Coughlin SR, Pleasure SJ. Identification of a transient
subpial neurogenic zone in the developing dentate gyrus and its regulation
by Cxcl12 and reelin signaling. Development. 2009;136:327–335.
30. Gorelova NA, Krivánek J, Bures J. Functional and metabolic correlates of
long series of cortical spreading depression waves in rats. Brain Res.
1987;404:379 –381.
31. Dohmen C, Sakowitz OW, Fabricius M, Bosche B, Reithmeier T,
Ernestus RI, Brinker G, Dreier JP, Woitzik J, Strong AJ, Graf R.
Spreading depolarizations occur in human ischemic stroke with high
incidence. Ann Neurol. 2008;63:720 –728.
32. Strong AJ, Hartings JA, Dreier JP. Cortical spreading depression: an
adverse but treatable factor in intensive care? Curr Opin Crit Care.
2007;13:126 –133.
33. Hartings JA, Rolli ML, Lu XC, Tortella FC. Delayed secondary phase of
peri-infarct depolarizations after focal cerebral ischemia: relation to
infarct growth and neuroprotection. J Neurosci. 2003;23:11602–11610.
34. Roelofs RF, Fischer DF, Houtman SH, Sluijs JA, Van HW, Van LFW,
Hol EM. Adult human subventricular, subgranular, and subpial zones
contain astrocytes with a specialized intermediate filament cytoskeleton.
Glia. 2005;52:289 –300.
35. Roegiers F, Jan YN. Asymmetric cell division. Curr Opin Cell Biol.
2004;16:195–205.
Induced Spreading Depression Evokes Cell Division of Astrocytes in the Subpial Zone,
Generating Neural Precursor-Like Cells and New Immature Neurons in the Adult
Cerebral Cortex
Jing-Hui Xue, Hiroji Yanamoto, Yukako Nakajo, Norimitsu Tohnai, Yoshikazu Nakano,
Takuya Hori, Koji Iihara and Susumu Miyamoto
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Stroke. 2009;40:e606-e613; originally published online September 24, 2009;
doi: 10.1161/STROKEAHA.109.560334
Stroke is published by the American Heart Association, 7272 Greenville Avenue, Dallas, TX 75231
Copyright © 2009 American Heart Association, Inc. All rights reserved.
Print ISSN: 0039-2499. Online ISSN: 1524-4628
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http://stroke.ahajournals.org/content/40/11/e606
An erratum has been published regarding this article. Please see the attached page for:
/content/41/7/e511.full.pdf
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Correction
In the article “Induced Spreading Depression Evokes Cell Division of Astrocytes in the Subpial
Zone, Generating Neural Precursor-Like Cells and New Immature Neurons in the Adult Cerebral
Cortex” by Xue et al,1 there are several errors in Figure 4C. The bars representing the density of
Vimentin positive cells at SPZ (the left bar in each group) are missing from day 1 to day 12 (ie,
all bars for the cell density at SPZ). The corrected Figure 4C can be seen below. The authors regret
this error.
The corrected version can be viewed online at http://stroke.ahajournals.org/cgi/content/full/40/11/e606.
1
[Correction for Vol 40, Number 11, November 2009. Pages e606 — e613.]
(Stroke. 2010;41:e511.)
© 2010 American Heart Association, Inc.
Stroke is available at http://stroke.ahajournals.org
DOI: 10.1161/STR.0b013e3181e9c433
e511