Experimental Neurology 226 (2010) 90–99

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Experimental Neurology
j o u r n a l h o m e p a g e : w w w. e l s e v i e r. c o m / l o c a t e / y e x n r

Increased subventricular zone-derived cortical neurogenesis after ischemic lesion

Maria Kreuzberg a,1, Evgeny Kanov a,b,1, Oleg Timofeev b, Markus Schwaninger c,
Hannah Monyer a, Konstantin Khodosevich a,⁎
a
Department of Clinical Neurobiology, Interdisciplinary Center for Neurosciences, Im Neuenheimer Feld 364, 69120 Heidelberg, Germany
b
Trans-Technologies, Ltd, St. Petersburg, Pesochny settl., Leningradskaya ul., 70/4, build. 6, Russia
c
Institute of Pharmacology, University of Heidelberg, Im Neuenheimer Feld 366, 69120 Heidelberg, Germany

a r t i c l e i n f o a b s t r a c t

Article history: In adult rodents stroke enhances neurogenesis resulting in the addition of neurons to forebrain regions such
Received 31 March 2010 as striatum or cortex where postnatal neurogenesis under normal conditions plays a negligible role. In the
Revised 3 August 2010 cortex, new neurons are generated either from local cortical precursors that are activated by stroke or from
Accepted 7 August 2010
precursors residing in the subventricular zone (SVZ) of lateral ventricles that under normal conditions
Available online 14 August 2010
supply neuroblasts by and large only for the olfactory bulb. In this study we used 5HT3A-EGFP transgenic
Keywords:
mice in which all neuroblasts originating in the SVZ are EGFP-labeled. We induced stroke in these mice and
Stroke by combination of EGFP detection with BrdU injections we labeled all post-stroke-generated SVZ-derived
SVZ neuroblasts. We showed an increase in SVZ-derived neuroblasts 14 and 35 days after stroke in the ipsilateral
Neurogenesis hemisphere. Post-stroke-generated SVZ-derived neuroblasts migrated to the cortex and survived for at least
Cortex 35 days representing 2% of BrdU-positive cells in peri-infarct area where they differentiate into mature
Neuroblasts neurons. Thus, stroke enhances SVZ neurogenesis and attracts newborn neurons to the injury zone.
© 2010 Elsevier Inc. All rights reserved.

Introduction neurogenic regions and promote migration of neuroblasts to the

Stroke, one of the most common causes of death and disability in
adulthood, is triggered by occlusion of a cerebral artery leading to
brain infarction. The resulting functional neurological deficits are
mainly due to the subsequent massive loss of neurons, suggesting that
cell replacement therapy might reduce the long-term consequences in
stroke patients.
There is an increasing body of evidence that stroke, which
experimentally can be induced in rats and mice by transient or
permanent middle cerebral artery occlusion, can trigger neurogenesis
in the subventricular zone (SVZ) of the lateral ventricle and the
subgranular zone (SGZ) of the dentate gyrus (Arvidsson et al., 2002;
Dempsey et al., 2003; Jin et al., 2001; Komitova et al., 2005; Parent
et al., 2002; Zhang et al., 2001), reviewed in (Wiltrout et al., 2007).
Under physiological conditions, neuroblasts generated in the SVZ
migrate tangentially along the rostral migratory stream (RMS) to the
olfactory bulb where they continue to migrate radially to reach their
final destination and differentiate into GABAergic periglomerular and
granule neurons (Ghashghaei et al., 2007; Khodosevich et al., 2009;
Lledo et al., 2006). Neurogenesis in the SGZ gives rise to glutamatergic
granule cells in the dentate gyrus (Abrous et al., 2005; Kempermann
et al., 2004). Ischemic brain injury can increase neurogenesis in both
⁎ Corresponding author. Fax: + 49 6221 561397.
E-mail address: khodosevich@urz.uni-hd.de (K. Khodosevich).
1
Equal contribution.
ischemic boundary in the cortex and striatum (Arvidsson et al., 2002;
Jin et al., 2001; Parent et al., 2002). In the striatum it has been
described that these newborn neuroblasts differentiate into GABAer-
gic medium spiny neurons or calretinin-positive GABAergic aspiny
inhibitory interneurons (Arvidsson et al., 2002; Hou et al., 2008; Liu et
al., 2009; Parent et al., 2002; Yang et al., 2008) and that the latter are
integrated into neuronal networks receiving synaptic input and firing
action potentials (Hou et al., 2008; Yamashita et al., 2006). In contrast,
relatively little is known about the fate of SVZ-derived neuroblasts
that are directed to the cortex following cortical stroke.
In the present study we analyzed the contribution of SVZ
progenitor cells to cortical neurogenesis in a cortex-specific stroke
model caused by middle cerebral artery occlusion (MCAO) using
5HT3A-EGFP transgenic mice, where EGFP is expressed under
promoter of serotonin receptor 5HT3A (Inta et al., 2008). In these
mice all SVZ-derived doublecortin-positive cells (=neuroblasts) are
EGFP-labeled. Upon maturation EGFP expression decreases in the
majority of neurons, but persists in those neurons, that normally
express the 5HT3A receptor (e.g., periglomerular cells in olfactory
bulb or layer 1 cortical interneurons). In addition, EGFP never co-
localizes with glial markers. Thus, by combining EGFP detection and
BrdU injections we were able to follow the migratory routes of post-
stroke-generated SVZ-derived neuroblasts. We found an increase in
post-stroke-generated neuroblasts in the SVZ of ischemic mice in both
the ipsilateral and contralateral hemisphere. However, while the
increase in neuroblast generation in the ipsilateral SVZ could be

0014-4886/$ – see front matter © 2010 Elsevier Inc. All rights reserved.
doi:10.1016/j.expneurol.2010.08.006
 M. Kreuzberg et al. / Experimental Neurology 226 (2010) 90–99
detected already 14 days after stroke, in the contralateral SVZ it was
observed only 35 days after stroke. In the peri-ischemic zone, 1% and
2% of all BrdU-positive cells were EGFP/BrdU double-positive
neuroblasts/neurons 14 days and 35 days after stroke, respectively.
In the cortex the majority of EGFP/BrdU double-positive neuroblasts
survived for at least 35 days after stroke. Many of them expressed
mature neuronal marker NeuN and were not labeled by apoptotic
marker activated caspase-3. In the frontal cortex, a portion of post-
stroke generated SVZ-derived neurons co-expressed calretinin, a
marker for a GABAergic interneuron subclass.
Materials and methods
Animals
For all our experiments, we used 5HT3A-EGFP transgenic mice
(Inta et al., 2008). All procedures with animals were performed
according to the Heidelberg University Animal Care Committee.
Materials and reagents
The following antibodies were used in the study: rabbit anti-EGFP
(1:10000; Molecular Probes, USA), chicken anti-EGFP (1:1500;
Abcam, UK), mouse anti-calretinin (1:1000; Swant, Switzerland),
rabbit anti-calretinin (1:1500; Swant, Switzerland), rabbit anti-
activated caspase-3 (1:1000; R&D Biosystems, USA), rat anti-BrdU
(1:500; Accurate, USA), mouse anti-NeuN (1:500; BD Biosciences,
USA), goat anti-doublecortin (1:500; Santa Cruz, Germany), mouse
anti-GFAP (1:4000; Sigma-Aldrich, Germany), goat anti-Olig2 (1:500;
R&D Biosystems, USA), mouse anti-Mash1 (1:500; BD Biosciences,
USA), rabbit anti-nestin (1:500; Abcam, UK), mouse anti-PSA-NCAM
(1:1000, Millipore, USA), Cy3-conjugated goat anti-rabbit, donkey
anti-goat and goat anti-rat (1:1000; Jackson Immuno, Germany), Cy5-
conjugated goat anti-rat, goat anti-mouse, donkey anti-goat and goat
anti-rabbit (1:1000; Jackson Immuno, Germany), Alexa 488-conju-
gated goat anti-chicken and goat anti-rabbit (1:1000; Molecular
Probes, USA).
Middle cerebral artery occlusion (MCAO)
For MCAO, 16–18 weeks old male 5HT3A-EGFP transgenic animals
(Inta et al., 2008) were used. MCAO was performed as previously
described (Zhang et al., 2005). Briefly, mice were anesthetized by
intraperitoneal injection of 150–200 μl 2.5% avertin solution per 10 g
body weight. A skin incision was made between the left ear and the
orbit, and the temporal muscle was removed by electrical coagulation
(Erbe, Germany). The skull was opened using a driller and the middle
cerebral artery was occluded by electrical coagulation. The wound
was closed with a skin suture. During surgery animals were kept at
body temperature using a heating pad. Only those animals that had
the similar extent of brain damage were used for the analysis.
BrdU application and dissection of brains
On days 1–6 after MCAO, mice obtained BrdU (Sigma-Aldrich
GmbH, Germany) to label dividing cells. BrdU was administered at a
concentration of 50 μg per g of body weight twice a day by
intraperitoneal injections. One or 4 weeks after the last BrdU
injection, animals were deeply anesthetized with 2.5% avertin
solution and perfused with 4% PFA. Brains were dissected and
postfixated with 4% PFA overnight at 4 °C.
Immunohistochemistry
Fifty-μm-thick sections were made using a Leica VT100S vibratome
(Leica Microsystems GmbH, Germany). Sections were washed in PBS,
91
incubated for 1 h with 5% normal goat serum (NGS) or 5% BSA in 0.1%
Triton-X-100 and subsequently overnight at 4 °C with primary
antibodies. After washing steps with PBS, sections were incubated
for 2 h with the respective secondary antibodies and mounted.
For BrdU double or triple labeling sections were washed in PBS and
incubated with 1 N HCl for 45 min followed by neutralization in
10 mM Tris–HCl pH 8.5 for 15 min. Sections were washed in PBS,
incubated for 1 h with 5% BSA in 0.2% Triton X-100 and subsequently
overnight at 4 °C with primary anti-BrdU antibodies. After washing
steps with PBS, sections were incubated for 2 h with the respective
secondary antibodies.
Sections were mounted on microscopy glass slides using Mowiol
(Sigma-Aldrich, Germany) and were analyzed with Olympus BX51TF
fluorescent (Olympus, Germany) and Zeiss LSM510 confocal micro-
scopes (Carl Zeiss, Germany).
Statistical analysis
The size of the areas containing EGFP-positive neuroblasts/
neurons in the subventricular zone and rostral migratory stream
was measured using ImageJ software and statistically analyzed.
Images were analyzed on every 3rd section through the whole SVZ
(13 sections for each brain, 7–9 brains for each condition) at high
magnification. All green fluorescent cells (up to individual neuro-
blasts) were outlined in ImageJ and included into analysis. If data
were not normally distributed according to d'Agostino and Shapiro–
Wilk tests, we used Kruskal–Wallis test for multiple comparisons and
Mann–Whitney test with Bonferroni correction for comparison
between single groups. For normally distributed data t-test was
used. Differences were considered significant at p b 0.05. All values in
graphs are mean ± standard deviation. For the expression analysis of
BrdU, BrdU/EGFP, BrdU/EGFP/activated caspase-3, BrdU/EGFP/Cr
and BrdU/EGFP/NeuN, at least 5 brains for each condition were used
and cell count was performed on 5–12 coronal sections in peri-infarct
area. The peri-infarct area was defined as a 300-μm brain region
surrounding the ischemic scar.
Results
Generation of SVZ neuroblasts is enhanced after ischemic stroke
Although several studies demonstrated an increase in SVZ
neurogenesis after ischemic stroke, they did not directly estimate
the increase in SVZ-derived neuroblasts in the cortex (Arvidsson et al.,
2002; Jiang et al., 2001; Jin et al., 2003; Leker et al., 2007; Parent et al.,
2002; Zhang et al., 2006, 2001; Ziv et al., 2007). We investigated
cortical neurogenesis after stroke combining BrdU injections and
EGFP detection in SVZ-derived neurons. BrdU injections allow the
labeling of proliferating cells and their descendants. However, since
the vast majority of BrdU-labeled cells in the ischemic zone are
microglia and astrocytes (Wang et al., 2004), the study of neurogen-
esis requires a combinatorial approach using in addition to BrdU
appropriate neuroblast markers. Here we utilized 5HT3A-EGFP mice,
thus profiting from the EGFP expression in all neuroblasts originating
in the SVZ (Inta et al., 2008) and investigated the extent of SVZ
response to stroke.
We injected 5HT3A-EGFP mice with BrdU on days 1–6 after stroke
and analyzed post-stroke neurogenesis in the SVZ 1 or 4 weeks after
the last BrdU injection (Fig. 1A). Fourteen days after stroke, the area of
EGFP-positive cells (neuroblasts) in the SVZ and RMS was signifi-
cantly increased in the ischemic hemisphere compared to the non-
ischemic hemisphere or sham-operated controls (7–9 animals per
group) (Fig. 1B–D, for confocal images of EGFP/BrdU cells see Fig. 4).
We observed an increase in SVZ neuroblast production in response to
stroke in both RMS and SVZ (Fig. 1C, D). Neuroblast generation in the
non-ischemic hemisphere was also slightly enhanced (Fig. 1B–D).
92 M. Kreuzberg et al. / Experimental Neurology 226 (2010) 90–99

Fig. 1. SVZ neurogenesis after cerebral ischemia in 5HT3A-EGFP mice. (A) Scheme of experiment. BrdU injections started 1 day after MCAO and continued for the 6 following days.
The analysis was performed 7 and 28 days after the last BrdU injection. (B–D) 14 days post-ischemia there was an increase in neuroblasts in the ischemic hemisphere in comparison
to the non-ischemic hemisphere and sham-operated animals. There was also a slight increase in the number of neuroblasts in the non-ischemic hemisphere in comparison to sham-
operated animals (*p b 0.001). (E–G) 35 days after MCAO, the number of neuroblast in the ischemic hemisphere was comparable to that at 14 days after MCAO, while neuroblast
number in the non-ischemic hemisphere increased significantly and almost reached that of the ischemic hemisphere (*p b 0.001). Scale bars—100 μm.

Five weeks after stroke, the area of EGFP-positive neuroblasts in
both SVZ and RMS was still significantly larger in the ischemic
hemisphere compared to that in sham-operated mice (Fig. 1E–G, for
confocal images of EGFP/BrdU cells see Fig. 4). In addition, the area of
EGFP-positive neuroblasts in the non-ischemic hemisphere was
also significantly larger compared to that in sham operated mice
(Fig. 1E–G). Interestingly, while the area of SVZ-derived neuroblasts
in the ischemic hemisphere was similar in size at 14 and 35 days after
stroke, in the non-ischemic hemisphere the area of SVZ-derived
neuroblasts was larger after 35 days compared to the earlier time-
point at 14 days post stroke (Fig. 1C, D and F, G, respectively). At the
later time-point the area of SVZ-derived neuroblasts in the non-
ischemic hemisphere was almost comparable in size with the
corresponding area in the ischemic hemisphere (Fig. 1F, G).
 M. Kreuzberg et al. / Experimental Neurology 226 (2010) 90–99
Neuroblasts produced in SVZ after stroke migrate and are retained in the
injured hemisphere
Two weeks after stroke we observed a stream of neuroblasts
descending from the SVZ into the ischemic area (Fig. 2A, B). In the
“ischemic” stream, all EGFP-positive cells co-expressed PSA-NCAM
and DCX (doublecortin), neuroblast markers (Fig. 2B1 and B2,
respectively). Concomitantly, in the SVZ and RMS there was 100%
co-localization between EGFP and DCX cells (Fig. 3A). Also, all EGFP/
BrdU-double positive cells in peri-infarct area co-expressed DCX
2 weeks, but not 5 weeks after stroke (data not shown). However, at
both time-points analyzed, EGFP-positive cells were never co-labeled
with Mash1 and nestin (Mash1 is a marker of transit-amplifying
precursors and nestin is a marker for neural stem cells and transit-
amplifying precursors in SVZ (Abrous et al., 2005; Lledo et al., 2006),
Fig. 3B and C, respectively), as well as with glial markers - GFAP
(astrocytic marker, Fig. 3D) and Olig2 (oligodendrocytic marker,
Fig. 3E). In Fig. 3F we have summarized the expression of EGFP in
5HT3A-EGFP transgenic mouse brain using data from this study and
from (Inta et al., 2008; Vucurovic et al., 2010). Note that EGFP is
expressed in practically all neuroblasts born in caudal ganglionic
eminence and postnatal SVZ (Inta et al., 2008), while after maturation
EGFP expression ceases in the majority of neurons.
We next analyzed the extent of post-stroke-generated neuroblast
migration to the injured cortex and whether they survive after several
weeks. BrdU incorporation in EGFP-positive cells of 5HT3A-EGFP mice
provides direct evidence that they were generated after stroke. Two
weeks after MCAO there were a number of post-stroke-generated SVZ-
derived neuroblasts in the peri-infarct area (Fig. 4A, B, B'). The majority
of EGFP/BrdU-double positive cells had only one process (Fig. 4B') and
in total these cells accounted for around 1% of BrdU-positive cells in the
peri-infarct area (Fig. 4D) [n = 5, 5 sections for each brain (every 6th
section of infarcted cortex), BrdU-positive cell count = 3811 ± 752,
BrdU/EGFP-double-positive cell count = 58 ± 14].
Thirty-five days after MCAO there were still a significant number of
EGFP/BrdU-double-positive cells in peri-infarct area—around 2% of
BrdU-positive cells (Fig. 4C, C', D) [n = 6, 5 sections for each brain (every
6th section of infarcted cortex), BrdU-positive cell count = 2572 ± 774,
Fig. 2. SVZ neuroblasts migrate to ischemic zone. (A–B) A stream of EGFP-positive SVZ neuroblasts migrating to the ischemic zone 2 weeks after MCAO. Cells in the stream express
neuroblast markers—PSA-NCAM (B1) and DCX (B2). In (A) white line borders the ischemic scar. Scale bars: in (A) 100 μm, in (B) 50 μm.
93
BrdU/EGFP-double-positive cell count = 53± 29]. However, since the
number of BrdU-positive cells was dramatically decreased 35 days after
MCAO relative to 14 days after MCAO (Fig. 4C and B, respectively), the
absolute number of EGFP/BrdU-double positive cells 35 days after
MCAO was only slightly lower than 14 days after MCAO.
Many post-stroke-generated SVZ-derived cortical neurons do not die by
apoptosis
Previous studies described that many neurons generated in the
cortex following stroke did not survive, and indicated that the
neurons died, most probably by caspase-dependent apoptosis
(Arvidsson et al., 2002; Thored et al., 2007). Hence, we examined
the survival of post-stroke born SVZ-derived neurons that migrated to
the peri-infarct area by immunolabeling with apoptotic markers. Of all
EGFP/BrdU double-positive cells, only 5% and 3% were also positive
for activated caspase-3, at 14 and 35 days after MCAO, respectively
(Fig. 5A–D) [for 14 days n = 5, 10 sections for each brain (every 3rd
section of infarcted cortex), BrdU/EGFP cells = 112 ± 26, BrdU/EGFP/
caspase-3 cells = 6 ± 4; for 35 days n = 5, 10 sections for each brain
(every 3rd section of infarcted cortex), BrdU/EGFP cells = 102 ± 59,
BrdU/EGFP/caspase-3 cells = 4 ± 2]. Thus, many of SVZ-derived
neurons do not die via apoptosis and survive in the peri-infarct area
at least 5 weeks after stroke (note that in experiments shown in Fig. 5
we used twice more brain sections in comparison to Fig. 3 experi-
ments, which resulted in more BrdU/EGFP cell counts).
Post-stroke-born SVZ-derived cortical neurons express markers of
mature cells
Finally, we analyzed whether neuroblasts generated in the SVZ
after stroke mature to cortical interneurons. Thirty-five days after
MCAO only 50% of EGFP/BrdU cells in the cortex were NeuN-positive
(Fig. 6A, B). However, this very likely is an underestimation of cortical
post-stroke-generated SVZ-derived neurons because of two factors:
first, in 5HT3A-EGFP mice a large fraction of postnatally generated
neurons (granule cells of the olfactory bulb) loses EGFP expression
upon maturation (data not shown) and second, the majority of SVZ-
94 M. Kreuzberg et al. / Experimental Neurology 226 (2010) 90–99

Fig. 3. Characterization of EGFP-positive cells in the ischemic brain. All EGFP-positive cells in SVZ co-express neuroblast marker DCX (A), but never express transit-amplifying
precursor marker Mash1 (B) or neural stem cell marker nestin (C). (D) EGFP-positive cells in the peri-infarct area do not express astrocytic marker GFAP. (E) EGFP-positive cells in
the peri-infarct area do not express oligodendrocytic marker Olig2. Note that overview images in (D) and (E) are projections of 5 stacks of 2.5-μm width while single cell confocal 3D
images are one 2.5-μm stack. In (E) white line borders the ischemic scar. (F) A scheme of EGFP expression in the brain of 5HT3A-EGFP mice relative to different stages of neuronal
differentiation produced using data obtained in this work as well as in (Inta et al., 2008; Vucurovic et al., 2010). Note that a small proportion of 5HT3A-EGFP neuroblasts continue to
express EGFP when mature. Scale bars—50 μm.

derived cortical neurons generated postnatally under normal condi- population in lower cortical layers which comprises the majority of
tions do not express NeuN, but express the interneuronal marker postnatally born cortical neurons—these EGFP/BrdU/Cr + neurons do
calretinin (Cr) [in (Inta et al., 2008) see EGFP/BrdU/Cr + neuronal not express NeuN (Khodosevich, Monyer, unpublished observation)].
 M. Kreuzberg et al. / Experimental Neurology 226 (2010) 90–99 95

Fig. 4. Post-stroke-generated SVZ-derived neuroblasts/neurons in the cortex. (A) EGFP/BrdU double-positive neuroblasts in the ipsilateral hemisphere migrate beyond the common
migratory pathways to the infarcted cortex. Arrows indicate double-positive cells. (B), (B') and (C), (C') post-stroke-generated SVZ-derived neuroblasts/neurons in the cortex
14 days and 35 days after MCAO, respectively. Arrowheads indicate double-positive cells. (D) Percentage of EGFP/BrdU double-positive cells relative to all BrdU-positive cells in the
peri-infarct area 14 and 35 days after MCAO. Scale bars: in (A) 50 μm, in (B) and (C) 100 μm, in (B') and (C') 20 μm.

The lack of NeuN expression has been reported previously for neurons
in several brain regions, e.g. cerebellum, olfactory bulb, substantia
nigra (Cannon and Greenamyre, 2009; Mullen et al., 1992; Weyer and
Schilling, 2003).
To assess the maturation of post-stroke-generated SVZ-derived
neurons, we analyzed the population of neurons in 5HT3A-EGFP
transgenic mice that still express EGFP when mature. Using 5HT3A-
EGFP mice, we have previously shown that upon maturation
postnatally SVZ-generated neurons in deep cortical layers of the
frontal cortex (Inta et al., 2008) express the interneuronal marker Cr.
Therefore we investigated the fate of EGFP-positive neuroblasts after
cerebral ischemia and focused on the population of Cr-positive
interneurons in deep frontal cortex layers. We found that the number
of triple-positive EGFP/BrdU/Cr interneurons in the frontal cortex and
the nearby RMS was much higher in the ischemic hemisphere in
comparison to the non-ischemic hemisphere 35 days after MCAO
(Fig. 7A–E) [n = 8, 12 sections for each brain (every 3rd section of
frontal cortex), BrdU/EGFP/Cr cells in ischemic hemisphere= 28 ± 14,
BrdU/EGFP/Cr cells in non-ischemic hemisphere = 8 ± 4]. Thus, a
portion of SVZ-derived neuroblasts, generated after stroke, migrates
to the frontal cortex and differentiates into Cr-positive interneurons.
Discussion
In the present study we investigated post-stroke neurogenesis and
its contribution to the generation of neurons in the cortex using the
MCAO model in 5HT3A-EGFP transgenic mice with EGFP expression in
SVZ-derived neurons. These transgenic mice in combination with
96 M. Kreuzberg et al. / Experimental Neurology 226 (2010) 90–99

Fig. 5. Apoptosis in cortical post-stroke-generated SVZ-derived neuroblasts/neurons. (A), (C) and (B), (D) Only few EGFP/BrdU double-positive cells are also activated caspase-3
(casp-3)-positive 14 and 35 days after stroke, respectively. Arrowheads—EGFP/BrdU double-positive, but activated caspase-3-negative cells. Arrow—EGFP/BrdU/activated caspase-3
triple-positive cell. Scale bars—20 μm.

BrdU injections provided us a novel tool to track SVZ-derived
neuroblasts produced during the first days after stroke. We found
that following stroke the neuroblast production in the SVZ is
enhanced in both the ipsilateral and contralateral hemisphere. We
showed that neuroblasts migrated to the cortex and could be detected
35 days after the infarct. The majority of SVZ-derived cortical
neuroblasts/neurons did not express apoptotic markers and many
cortical SVZ-derived neuroblasts, generated in response to the stroke,
differentiated into mature neurons expressing NeuN or calretinin.
Although many studies described cortical neurogenesis after
stroke (Arvidsson et al., 2002; Jiang et al., 2001; Jin et al., 2003;
Leker et al., 2007; Parent et al., 2002; Zhang et al., 2006, 2001; Ziv
et al., 2007), they did not address the extent of SVZ-derived
neuroblast recruitment to the cortex in response to stroke. To control

Fig. 6. Maturation of post-stroke-generated SVZ-derived neurons in ischemic brain. (A) and (B) EGFP/BrdU/NeuN triple-positive cells in the cortex 35 days after MCAO. Scale
bars—10 μm.
 M. Kreuzberg et al. / Experimental Neurology 226 (2010) 90–99 97

Fig. 7. Maturation of post-stroke-generated SVZ-derived neurons in frontal cortex of ischemic hemisphere. EGFP/BrdU and interneuronal marker calretinin (Cr) triple-positive
neurons in the contralateral (A) and the ipsilateral (B) to the stroke hemisphere (in the area adjacent to RMS). Note higher number of triple-positive cells in the ipsilateral
hemisphere. (C and D) Examples of EGFP/BrdU/Cr triple-positive cells in frontal cortex. (E) The number of EGFP/BrdU/Cr triple-positive cells in frontal cortex is dramatically
increased in the ipsilateral hemisphere compared to the contralateral side (*p b 0.005). Scale bars: in (A) and (B), 20 μm; in (C) and (D), 10 μm.
98 M. Kreuzberg et al. / Experimental Neurology 226 (2010) 90–99
both birth-dating and the origin of neurons, combined BrdU injections
with transgenic or viral labeling of SVZ-derived neuroblasts is
required. Such an approach was used by Burns et al. (2007) to trace
SVZ-derived neuroblasts in the striatum after stroke. The authors
found only few BrdU-positive cells that were produced in the SVZ,
which indicates only a small contribution of the SVZ to striatal post-
stroke neurogenesis. However, this can be an underestimation,
because tamoxifen-activated Cre recombination, which was used for
cell tracking, might not label all precursor cells since in some adult Cre
expressing strains the transgenic promoter is methylated and Cre
recombination is inhibited (see e.g. (Long and Rossi, 2009)). In our
study, we used transgenic mice expressing EGFP under the control of
the 5HT3A promoter that allowed the tracing of all neuroblasts
generated in the SVZ. We combined MCAO experiments with BrdU
injections, thus, labeling all EGFP-positive neuroblasts that were
produced in the SVZ in response to stroke. The contribution of the SVZ
to cell replacement in injured cortex was significant given that 1% of
BrdU-positive cells in the peri-infarct area were also EGFP-positive, i.e.
neuroblasts/neurons, 14 days after MCAO and 2% of BrdU-positive
cells were EGFP-positive 35 days after MCAO (Fig. 4D). The increase
that at a first glance might be puzzling can be accounted for by the fact
that BrdU labeling also includes a number of astrocytes and microglia,
many of which do not survive (Wang et al., 2004). The absolute
number of EGFP/BrdU double-positive cells in post-ischemic cortex
was slightly higher at the earlier time point.
We showed in a previous study (Inta et al., 2008) and confirmed it
here that all EGFP-positive cells generated in the SVZ co-express
neuroblast marker DCX. Thus, all cells that are migrating to the
ischemic zone are neuroblasts. The reactivation of 5HT3A promoter,
and subsequent labeling of non-SVZ-derived cells, in some local
precursors is very unlikely due to several reasons. First, following
stroke we always observed a stream of migrating EGFP-positive
neuroblasts starting in the SVZ and ending in the peri-infarct area (see
Fig. 2A, B). Second, local cortical precursors generate many glial cells
in response to stroke, while EGFP-positive cells never co-express glial
markers. And finally, SVZ precursors and adult cortical precursors are
quite different cells, and in 5HT3A-EGFP mice only those cells that
were born in postnatal SVZ and prenatal caudal ganglionic eminence
are labeled (Inta et al., 2008).
In the striatum around 80% of post-stroke-generated neurons die
between 2 and 6 weeks after stroke (Arvidsson et al., 2002). This
massive cell death is most likely caspase-dependent since inhibition
of caspase function remarkably increased neuronal survival in the
striatum (Thored et al., 2007). We showed in this study that many of
cortical post-stroke-generated neurons did not express activated
caspase-3 and survived at least 5 weeks after stroke.
We demonstrated that at least half of the BrdU/EGFP cells 35 days
after MCAO were mature neurons as they exhibited expression of the
neuronal marker NeuN. However, it is possible that in these transgenic
mice we underestimated the number of SVZ-derived mature neurons
since the majority of cortical neurons generated postnatally in SVZ
under non-pathological conditions are Cr-positive, but NeuN-negative
[see above and (Inta et al., 2008)]. Also, in other brain regions such as the
olfactory bulb we could observe that 5HT3A-EGFP-positive neuroblasts
upon differentiation lose EGFP expression (data not shown). Interest-
ingly, Leker et al (Leker et al., 2007) showed in the distal MCAO rat
model that 3 months after MCAO 12% of BrdU-positive cells were also
NeuN-positive. This discrepancy might result from species differences
(rat versus mouse) or different time points of analysis (90 versus
35 days after MCAO). Also, it is possible that the majority of post-stroke-
generated neurons are derived from local cortical precursors rather than
SVZ cells, and, thus, omitted from our analysis.
Previously we have shown that many Cr-positive interneurons in
the frontal cortex are generated postnatally from SVZ precursors (Inta
et al., 2008). This study reveals, that the generation of these Cr-
positive interneurons is stimulated after stroke resulting in an
increased number of newborn Cr-positive interneurons in the
ipsilateral hemisphere. However, we cannot clarify whether this
increase in Cr-positive interneurons is a result of the overall increased
proliferation in the ipsilateral SVZ only or whether in addition certain
regional cues might be present in the cortex of the ipsilateral
hemisphere, facilitating the integration of newborn neuroblasts into
cortical circuits.
According to our data there are 50–100 Cr-positive cells per
hemisphere that are generated in SVZ during the first days following
stroke (28 ± 14 in every third section per ischemic hemisphere).
Although the number of cells is not that high, activation of neuronal
generation after injury can reflect the plasticity of adult/mature brain
and its adaptation to external stimuli. These Cr-positive cells migrate
to the cortical region that is far away from the injury zone and might
represent an intrinsic brain attempt to recover from the injury.
However, it remains to be investigated whether these post-stroke
generated cells are integrated in the local circuits.
It was shown in several studies that cell proliferation in the
ipsilateral SVZ was increased already 1 to 2 weeks after stroke and
returned to baseline 6 weeks after stroke (Arvidsson et al., 2002;
Zhang R.L. et al., 2004; Zhang R. et al., 2004). In the current study, the
number of neuroblasts in the ipsilateral SVZ was twice that of the
contralateral SVZ 2 weeks after stroke. However, 5 weeks after the
stroke, the number of neuroblasts in the contralateral SVZ was almost
comparable to that of the ipsilateral SVZ. Although Leker et al. (2007)
showed an increase in newborn cells in the contralateral hemisphere
30 days after stroke, the effect was milder than in the ipsilateral
hemisphere. In our study, the area of neuroblasts in both ipsilateral
and contralateral SVZ was approximately the same and 2 times larger
compared to sham-operated mice. Thus, we showed that in the
hemisphere contralateral to stroke, the SVZ also reacts to brain injury,
but later than ipsilaterally. This might be due to the longer time
required for some factors to diffuse to the contralateral SVZ to trigger
an increase in neuroblast production. Alternatively, some regional
factors first influence the ipsilateral hemisphere and stimulate
neurogenesis and later systemic factors modulate the activity in the
whole brain.
In spite of major efforts to develop treatment following stroke, it
has remained one of the leading causes of death and disability in
adults. There is an increasing number of studies showing that
progenitor cells residing in the SVZ are potentially able to replace
the loss of nervous tissue after stroke. Thus, modulation of intrinsic
neurogenesis in SVZ following stroke is a promising approach aiming
at functional restoration of damaged brain circuits and increased
recovery after stroke.
Acknowledgments
This work was supported in part by the DFG (SFB488 grant), the
BMBF (RUS 09/B38), and European Union's Seventh Framework
Programme (FP7/2007-2013) under grant agreements 201024 and
202213 (European Stroke Network).
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