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Neuronal replacement from endogenous precursors in the adult brain after stroke.
Arvidsson, Andreas; Collin, Tove; Kirik, Deniz; Kokaia, Zaal; Lindvall, Olle
Published in:
Nature Medicine
DOI:
10.1038/nm747
2002
Link to publication
Citation for published version (APA):
Arvidsson, A., Collin, T., Kirik, D., Kokaia, Z., & Lindvall, O. (2002). Neuronal replacement from endogenous
precursors in the adult brain after stroke.
Nature Medicine
,
8
(9), 963-970. https://doi.org/10.1038/nm747
Total number of authors:
5
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NATURE MEDICINE • VOLUME 8 • NUMBER 9 • SEPTEMBER 2002 963
ARTICLES
The generation of new neurons in the adult brain is largely re-
stricted to two regions: the subventricular zone (SVZ) lining the
lateral ventricles, and the subgranular zone (SGZ) of the dentate
gyrus (DG)
1
. Additional neuronal progenitors reside in the fore-
brain parenchyma
2
. Brain insults such as cerebral ischemia and
epileptic seizures, which cause neuronal death, are accompanied
by increased neurogenesis in the SGZ (refs. 3–6) and SVZ (refs.
7,8), whereas it is unclear if new neurons are formed in other re-
gions
8,9
.
From the clinical perspective, a fundamental question is
whether the new neurons can migrate to the site of injury and re-
place neurons that have died. The evidence for such neuronal self-
repair in the adult brain is scarce. Magavi et al.
10
used targeted
apoptosis of cortical pyramidal neurons and found a small num-
ber of new neurons extending processes to the original target sites
in the thalamus. However, this lesion destroyed only the targeted
neurons without affecting the surrounding tissue. Whether neu-
ronal replacement from endogenous precursors occurs following
extensive lesions resembling human disease is unknown.
Here we investigated whether new neurons are formed in the
adult rat striatum after stroke induced by two hours of middle
cerebral artery occlusion (MCAO). This ischemic insult causes in-
farction in the striatum and parietal cortex. We show that stroke
leads to increased neurogenesis in the SVZ. New neurons migrate
into the damaged striatal area, where they express morphological
markers characteristic of most of the dead neurons, that is,
medium-sized spiny neurons
11
. In the intact brain, these neurons
project to the globus pallidus and substantia nigra and form part
of the basal ganglia motor circuitry.
Stroke leads to neurogenesis in damaged striatum
At five weeks after stroke, there was an almost complete loss of
cells immunoreactive for the neuron-specific marker NeuN in
the lateral and caudal parts of the ipsilateral striatum, whereas
the most medial and frontal parts were often spared. To label
dividing cells we used 5-bromo-2′-deoxyuridine (BrdU), a
thymidine analog that is incorporated into DNA during cell di-
vision. BrdU was injected twice daily during days 4–6 after
stroke or sham procedure. At 4 weeks after the last injection,
few BrdU-NeuN double-labeled, presumably newly formed
neurons were observed in the striatum ipsilateral to sham
surgery. Stroke gave rise to a 31-fold increase of the number of
BrdU-NeuN-labeled neurons in the ipsilateral striatum (Fig. 1a)
(n = 9 and 10 for MCAO and sham, respectively; P < 0.005,
Mann–Whitney U-test), whereas the number in the contralat-
eral striatum was similar to that in control (data not shown).
There were numerous BrdU-labeled cells not expressing NeuN
in the ischemic brain tissue, reflecting massive gliosis and in-
flammatory reaction
12,13
. Parts of the damaged striatal tissue
had often disintegrated and were not available for analysis.
Therefore, the stroke-induced increase of BrdU-NeuN double-
labeled cells was even more pronounced when cell densities
were compared between ischemic and sham-operated animals
(136.7 ± 66.7 and 0.8 ± 0.3 cells per mm
3
, respectively; P <
0.0005, Mann–Whitney U-test) (Fig. 1b). In the double-labeled
cells, the BrdU staining was strong and equally distributed
throughout the nucleus (Fig. 1c–n). Most BrdU-NeuN im-
munoreactive cells were localized within the damaged area, es-
pecially medially and dorsally. Also, in the seemingly
unaffected medial striatum, there was increased density of dou-
ble-labeled neurons (28.7 ± 12.2 cells per mm
3
; P < 0.05,
Mann–Whitney U-test) (Fig. 1b). We found no clearly BrdU-
NeuN double-labeled cells in or around the infarcted area of
the parietal cortex.
Neuronal replacement from endogenous precursors in the
adult brain after stroke
ANDREAS ARVIDSSON
1
, TOVE COLLIN
1
, DENIZ KIRIK
2
, ZAAL KOKAIA
1
& OLLE LINDVALL
1
1
Section of Restorative Neurology and
2
Neurobiology, Wallenberg Neuroscience Center,
Lund University Hospital, Lund, Sweden
Correspondence should be addressed to A.A.; email: andreas.arvidsson@neurol.lu.se
Z.K. and O.L. contributed equally to this study.
Published online: 5 August 2002, corrected online 30 August 2002 (details online); doi:10.1038/nm747
In the adult brain, new neurons are continuously generated in the subventricular zone and den-
tate gyrus, but it is unknown whether these neurons can replace those lost following damage or
disease. Here we show that stroke, caused by transient middle cerebral artery occlusion in adult
rats, leads to a marked increase of cell proliferation in the subventricular zone. Stroke-generated
new neurons, as well as neuroblasts probably already formed before the insult, migrate into the
severely damaged area of the striatum, where they express markers of developing and mature,
striatal medium-sized spiny neurons. Thus, stroke induces differentiation of new neurons into
the phenotype of most of the neurons destroyed by the ischemic lesion. Here we show that the
adult brain has the capacity for self-repair after insults causing extensive neuronal death. If the
new neurons are functional and their formation can be stimulated, a novel therapeutic strategy
might be developed for stroke in humans.
© 2002 Nature Publishing Group http://www.nature.com/naturemedicine
964 NATURE MEDICINE • VOLUME 8 • NUMBER 9 • SEPTEMBER 2002
ARTICLES
Stroke triggers proliferation and recruitment of neuroblasts
The new neurons could originate from either the SVZ or from
progenitors residing in the striatal parenchyma. We first investi-
gated whether the insult had increased cell proliferation in the
SVZ. Animals were given BrdU injections twice daily for two
weeks after stroke and were sacrificed one day thereafter. Both
the area covered by the BrdU-labeled cells and their number in
the SVZ were markedly increased on the ischemic as compared
with the contralateral side (area, 64,400 ± 7,000 and 42,600 ± 3,
000 µm
2
, P < 0.05; cell number, 639 ± 111 and 289 ± 36 cells per
section, P < 0.01, respectively; paired t-test, n = 9). In contrast, the
side difference in sham-operated rats was only marginal (area,
47,200 ± 2,200 and 48,500 ± 2,500 µm
2
, P > 0.05; cell number,
395 ± 15 and 342 ± 18 cells/section, P < 0.05, respectively; paired
t-test, n = 5).
To confirm that BrdU incorporation following stroke was due to
cell proliferation in the SVZ, we infused the antimitotic drug cyto-
sine-β-
D-arabinofuranoside (Ara-C)
14
during the 12-day period after
stroke and injected BrdU in parallel. Ara-C treatment inhibits cell
proliferation in the mouse SVZ (ref. 14). The MCAO gave rise to an
increase in SVZ size and BrdU
+
cell numbers in saline-treated ani-
mals at one day after the last BrdU injection, but these values were
markedly reduced by Ara-C treatment (area, 68,900 ± 6,500 and
30,300 ± 3,100 µm
2
; cell number, 1,027 ± 166 and 174 ± 36 cells per
section for saline and Ara-C treated animals, respectively; n = 5 and
8; P < 0.0001 for both comparisons; unpaired t-test) (Fig. 2a).
a
b
c d e
f
g
h
i
j
k
l
m
n
Fig. 1 Stroke-induced neurogenesis in the adult striatum. a and b,
Number (a) and density (b) of cells double-labeled with BrdU and NeuN in
the striatum ipsilateral to sham surgery or 2 h of MCAO followed by BrdU
injections at days 4–6 and 4 weeks of survival, as quantified with confocal
microscopy in six 20-µm brain sections. Data are means ± s.e.m.; *, P <
0.005 (a); *, P < 0.0005 (b) compared with sham surgery, Mann–Whitney
U-test. n = 10 and 9 for sham and MCAO, respectively. Densities in b are
shown separately for parts of the striatum undamaged by the ischemia
(MCAO intact) and for all striatal tissue (MCAO total). c–h, Confocal 3D re-
construction of neurons from the intact (c–e) and lesioned (f–h) parts of
the striatum ipsilateral to MCAO showing NeuN (c and f) or BrdU (d and g)
immunoreactivity separately or as a merged image (e and h).
Reconstructed orthogonal images are presented as viewed in the x–z (top)
and y–z (right) planes. i–k, Examples of striatal cells immunoreactive for
NeuN (i, green) and BrdU (j, red) and labeled with the nuclear stain DAPI
(k, blue). l–n, 10 consecutive 1-µm confocal images in z-dimension show-
ing NeuN (l) or BrdU (m) immunoreactivity separately or as merged im-
ages (n). Arrows indicate double-labeled cells. Scale bar, 15 µm.
© 2002 Nature Publishing Group http://www.nature.com/naturemedicine
NATURE MEDICINE • VOLUME 8 • NUMBER 9 • SEPTEMBER 2002 965
ARTICLES
To assess the role of cell proliferation in the generation of new
striatal neurons, we stained for a marker of migrating neuroblasts,
doublecortin (Dcx). In the adult brain, Dcx is expressed in the SVZ
and rostral migratory stream but only single cells are detected in
the striatum
15
. We found a similar pattern of expression on the
side contralateral to stroke in the saline-treated animals, whereas
Dcx-labeled cells were abundant on the ipsilateral side (Fig. 2b and
c). After Ara-C treatment, the number of BrdU-Dcx double-labeled
cells was markedly reduced in the ischemic striatum (P < 0.005,
unpaired t-test) (Fig. 2b–l), whereas there remained numerous
Dcx-stained, BrdU
–
cells (P > 0.05 (see Fig. 2b, unpaired t-test) (Fig.
2b and j–l). This finding indicates that most but not all of the new
striatal neurons had been generated through stroke-induced cell
proliferation in the SVZ or striatal parenchyma.
Stroke-generated neurons migrate from SVZ to damaged area
In the animals which were given BrdU injections for 14 days after
the insult and killed directly thereafter, large numbers of Dcx-im-
munoreactive neurons appeared to migrate from the SVZ laterally
and ventrally into the damaged area in the ischemic striatum (Fig.
3b). In contrast, both contralateral to the insult and in the rats
subjected to sham procedure, Dcx immunoreactivity was con-
fined to the SVZ and single striatal cells (Fig. 3a and c). No Dcx-im-
munoreactive cells were observed in or around the infarcted area
of the parietal cortex.
The stroke-generated Dcx
+
cells showed morphologies charac-
teristic of both migrating (elongated and leading processes) (Fig.
3d–f) and non-migrating cells (more symmetric with several
processes in different directions) (Fig. 3g–i). The elongated Dcx
+
cells sometimes formed aggregates resembling chains. Confocal
microscopy showed that 62% of the Dcx-immunoreactive neu-
rons were also labeled with BrdU. Unlike the BrdU-NeuN-dou-
ble-labeled cells, which were preferentially located in the
damaged area at 5 weeks, the BrdU-Dcx-stained cells detected at
2 weeks were distributed along a gradient from the SVZ into the
striatal parenchyma. Typically, more than half of these cells
were located up to 0.5 mm laterally from the SVZ, and their den-
sity then gradually tapered off. Scattered cells could be observed
up to approximately 2 mm from the SVZ. Most cells with migrat-
ing morphology had their leading processes directed away from
the SVZ.
Because the results of the Ara-C treatment suggested that about
half of the Dcx
+
BrdU
–
neurons were generated after the stroke, the
BrdU labeling probably underestimates the magnitude of striatal
neurogenesis. Also, in brain areas normally sustaining adult neu-
rogenesis, that is, DG and olfactory bulb, we found cells labeled
only with Dcx along with a high number of Dcx
+
cells colabeled
with BrdU (Fig. 3j–m). We therefore used Dcx as the main marker
of newly formed striatal neurons for the further characterization
of their phenotype during early development.
a b c d
e
f
g h
i
j
k
l
Fig. 2 Ara-C treatment inhibits proliferation in SVZ and
striatal neurogenesis after stroke. a and b, Number of BrdU
+
(a) and density of Dcx
+
BrdU
+
or Dcx
+
BrdU
–
cells (b) in the ip-
silateral SVZ (a) and striatum (b) at 12 d after 2 h MCAO
and saline () or Ara-C () infusion with concomitant BrdU
injections until perfusion. Data are means ± s.e.m.; *, P <
0.0001 compared with saline infusion, unpaired t-test. n =5
and 8 for saline and Ara-C, respectively. c–f, Overview of
Dcx (c and e) and BrdU (d and f) immunoreactivity in the
dorsomedial striatum ipsilateral to the insult in animals in-
fused with saline (c and d) or Ara-C (e and f) at 12 d after
MCAO. Note abundant Dcx
+
cells in the SVZ and dorsome-
dial part of the striatum of saline-infused animals. Also note
the pronounced decrease of Dcx-immunoreactivity in the
SVZ and striatum (e) and BrdU-immunoreactivity in the SVZ
(f) in the animals subjected to Ara-C infusions.LV, lateral
ventricle. g–l, Confocal images from the same plane illus-
trating Dcx (g and j) and BrdU (h and k) immunoreactivity
separately or as a merged image (i and l). Note double-la-
beling for Dcx and BrdU in saline- (g–i) but not in Ara-C-
(j–l) infused animals (compare i and l). Arrows indicate dou-
ble-labeled cells. Scale bar in c is 200 µm for c–f and 40 µm
for g–l.
© 2002 Nature Publishing Group http://www.nature.com/naturemedicine
966 NATURE MEDICINE • VOLUME 8 • NUMBER 9 • SEPTEMBER 2002
ARTICLES
New cells express markers of striatal medium spiny neurons
In order to determine whether the new neurons developed
striatal-specific characteristics, we first analyzed their expres-
sion of the transcription factor Meis2 at 2 weeks after stroke.
Meis2 is normally expressed in proliferating precursors that
differentiate into striatal neurons, but is also expressed in the
adult striatum where the expression closely resembles that of
the marker for medium-sized spiny neurons, DARPP-32 (ref.
16). Confocal microscopy showed that virtually all Dcx
+
cells
(96%) were Meis2
+
(Fig. 4a–c). The expression of Pbx proteins,
which are colocalized with Meis2 in developing striatal neu-
rons
16
, coincided with that of Meis2, and almost all Dcx
+
cells
(94%) (Fig. 4d–f) were also labeled with Pbx. We found simi-
larly high coexpression of Dcx and Hu (93%) (Fig. 4g–i), an
early neuronal marker
17,18
. There was also extensive colocaliza-
tion of BrdU with Meis2, Pbx or Hu in the SVZ and striatum
(Fig. 4j–r). In addition, Meis2 was expressed in Dcx
–
and BrdU
–
striatal neurons on the lesioned and non-lesioned side.
However, the Meis2 immunoreactivity was stronger in the
cells colabeled with BrdU or Dcx as compared with the other
striatal neurons. This finding is consistent with previous ob-
servations that Meis2 expression is higher in developing than
in mature striatal neurons
19
. We did not observe a single Dcx-
labeled cell that coexpressed markers of glial precursors or ma-
ture glia, that is as NG-2, vimentin and glial fibrillary acidic
protein (GFAP).
At two weeks after the stroke, 20% of Dcx
+
striatal cells also
expressed NeuN, indicating that some of the young cells had
already differentiated into mature neurons. This was substan-
tiated by the finding of cells double-labeled with BrdU and
NeuN in the same brains (Table 1). We then allowed an addi-
tional group of rats, subjected to the same regimen of BrdU
injections, to survive for 4 weeks following the last injection.
The number and density of BrdU-NeuN double-labeled cells
was 4.9- and 9.7-fold higher, respectively, in the ischemic
striatum of these animals as compared with those that had
been sacrificed directly following the last BrdU injection (P <
0.005 for both number and density, Mann–Whitney U-test,
a b
c
d
e
f
g
h
i
j
k
l
m
Fig. 3 Stroke-generated cells migrate from the subventricular zone into
the ischemic lesion. a and b, Overview of Dcx (green) and BrdU (red) im-
munoreactivity in the dorsomedial striatum contralateral (a) and ipsilateral
(b) to the insult at 2 wk after MCAO. On the contralateral side, Dcx im-
munoreactivity is restricted to the SVZ and isolated cells in the striatum (a),
whereas abundant Dcx
+
cells are present in the ipsilateral striatum (b), dis-
tributed in a density gradient from the SVZ, bordered dorsally by the cor-
pus callosum (CC) and medially by the lateral ventricle (LV). c–f, Confocal
microscopy images showing the boxed areas in a and b. c, A Dcx
+
BrdU
–
cell
with the morphology of a mature neuron located in the striatum contralat-
eral to MCAO. d, Densely clustered Dcx
+
cells in the SVZ, showing extensive
colocalization with BrdU immunoreactivity. A migrating Dcx-BrdU double-
labeled cell can be seen in the CC dorsal to the SVZ (arrow). e,A Dcx
+
cell
with the morphology of a migrating neuroblast double-labeled with BrdU.
f, Multiple Dcx-labeled cells with migratory or mature morphologies. Some
of the cells are double-labeled with BrdU (arrows). g–i, Confocal images of
two neurons with mature morphology from the striatum ipsilateral to
MCAO showing Dcx (g) or BrdU (h) immunoreactivity separately or as a
merged image (i). j–m, Overviews (j and k) and confocal images (l and m)
showing boxed areas in j and k, of Dcx and BrdU immunoreactivity in the
dentate gyrus (j and l) and olfactory bulb (k and m) ipsilateral to sham
surgery. There is extensive, but not complete, colocalization of Dcx and
BrdU immunoreactivity. Scale bar in a is 200 µm for a, b and k, 40 µm for
c–e, l and m, 50 µm g–I and 60 µm for j.
© 2002 Nature Publishing Group http://www.nature.com/naturemedicine
