European Journal of Neuroscience, Vol. 21, pp.

23972405, 2005

Federation of European Neuroscience Societies

Postischemic exercise attenuates whereas enriched

environment has certain enhancing effects on lesioninduced subventricular zone activation in the adult rat
Mila Komitova,1 Li Ru Zhao,2 Gunilla Gido,2 Barbro B. Johansson2 and Peter Eriksson1
1

The Arvid Carlsson Institute at the Institute of Clinical Neuroscience, Medicinaregatan 11, Box 432, 405 30 Goteborg, Sweden
Division for Experimental Brain Research, Department of Clinical Neuroscience, Lund University, Wallenberg Neuroscience
Center, Lund, Sweden

Keywords: middle cerebral artery occlusion, neural progenitor cell, neural stem cell, neurogenesis

Abstract
Experimental stroke increases cell proliferation and neurogenesis in the subventricular zone (SVZ) and in the dentate gyrus
subgranular zone (SGZ) in the adult mammalian brain. This study examined the effects of postischemic voluntary exercise
(running wheel) and environmental enrichment on the SVZ and SGZ 1 week after focal cortical ischemia in adult spontaneously
hypertensive rats. Immunohistochemical labeling was performed for incorporation of specific cell markers such as Ki67 and
5-bromodeoxyuridine (proliferating and newborn cells), terminal deoxynucleotidyl transferase-mediated dUTP in situ nick-end
labeling (apoptotic cells), Sox-2 and glial fibrillary acidic protein (neural stem and progenitor cells), polysialylated neural cell
adhesion molecule and doublecortin (neuroblasts). Postischemic exercise and environmental enrichment differentially modulated
SVZ cell genesis but lacked effects on the SGZ. Lesion-induced proliferation of neural stem progenitor cells and neuronal
precursors was attenuated in stroke runners without any effects on apoptosis or neuronal migration in the forebrain. Running
activity did not affect the SVZ in intact rats. In contrast to postischemic wheel running, postischemic environmental enrichment
did not have attenuating effects on the ipsilateral SVZ and increased proliferating putative neural stem cells and neuronal
precursors contralaterally. A significant functional improvement, assessed using a rotating pole, was observed only in the
postischemically enriched group and was likely due to other types of plasticity than neuronal replacement at this early time point.
It may be concluded that in contrast to enriched environment, exercise during the first postischemic week might be detrimental
for regenerative processes initiated in the SVZ after stroke.

Introduction
An ischemic brain lesion results in a substantial increase in the
proliferation of neural progenitor cells, subsequently leading to
increased neurogenesis in the two neurogenic regions in the adult
mammalian brain: the subgranular zone (SGZ) at the border
between the granule cell layer (GCL) and the polymorphic layer of
the dentate gyrus in the hippocampal formation, and the subventricular zone (SVZ) in the forebrain (Liu et al., 1998; Arvidsson
et al., 2001; Jin et al., 2001; Zhang et al., 2001; Komitova et al.,
2002; Kokaia & Lindvall, 2003). This endogenous response is
particularly important to study because newborn neurons from the
SVZ have been shown to be recruited to nearby infarcted areas in
the cortex and the striatum and can start to express mature and
region-specic markers (Arvidsson et al., 2002; Parent et al., 2002;
Jin et al., 2003; Zhang et al., 2004).
Two environmental interventions that have been shown to have
benecial effects on hippocampal neurogenesis in intact rats and mice
are environmental enrichment and exercise (Kempermann et al., 1997;
Nilsson et al., 1999; van Praag et al., 1999; Farmer et al., 2004;

Correspondence: Dr M. Komitova, as above.

E-mail: mila.komitova@neuro.gu.se
Received 3 October 2004, revised 22 February 2005, accepted 24 February 2005

doi:10.1111/j.1460-9568.2005.04072.x

Persson et al., 2004). One study has, however, shown that exercise and
enriched environment lack effects on basal levels of SVZ cell genesis
in mice (Brown et al., 2003).
The postischemic effects of these environmental interventions on
cell genesis in the germinal niches have only recently begun to be
elucidated. In a previous study in which rats with a cortical infarct
were given the thymidine analog bromodeoxyuridine (BrdU) and
allowed to survive for 5 weeks after the surgery, we found that
postischemic environmental enrichment lacked effects on the total
number of surviving newborn cells and new neurons in the
hippocampus but rather increased gliogenesis and normalized the
ratio between newborn astrocytes and neurons that was perturbed by
the insult (Komitova et al., 2002).
The aims of this study were several. We wanted to study possible
early effects on hippocampal cell proliferation and early neurogenesis,
questions that were not addressed in our previous study. Moreover we
wanted to extend our studies to also include the SVZ. Thirdly, we
wanted to compare the effects of postischemic environmental
enrichment with those of postischemic voluntary exercise on the
germinal niches because earlier comparative studies of these environmental interventions have demonstrated differential behavioral outcome and gene expression data (Johansson & Ohlsson, 1996; Risedal
et al., 2002; Dahlqvist et al., 2003).

2398 M. Komitova et al.

Materials and methods

Immunohistochemistry

Animals, surgery and housing conditions

Immunohistochemistry was performed as previously described

(Komitova et al., 2002). Briey, sections were incubated in blocking
solution for 30 min (3% donkey serum and 0.1% Triton X-100 in Trisbuffered saline, TBS: 0.08 m Trizma-HCl, 0.016 m Trizma-Base,
0.15 m NaCl, pH 7.5) and then incubated with primary antibody in
blocking solution for 16 h at 4
C. After rinsing with TBS and
incubating in blocking solution for 15 min, sections were incubated
for 2 h at room temperature with uorochrome-conjugated secondary
antibody or biotinylated antibody, the latter reacted with avidinperoxidase for 1 h (ABC-kit, Vectastain Elite, Vector Laboratories)
followed by detection solution (26.5 mg mL diaminobenzidine,
0.01% H2O2, 0.04% NiCl). In order to detect BrdU, the halogenated
pyrimidine was exposed through DNA denaturation before incubation
in primary antibody. This was achieved by treating the sections for 2 h
at 65
C in sodium citrate buffer (0.03 m NaCl, 0.3 m sodium citrate,
pH 7) containing 50% formamide. The sections were then rinsed in
sodium citrate buffer for 15 min, incubated in 2 m HCl for 30 min at
37
C, rinsed in 0.1 m borate buffer (pH 8.5) for 10 min followed by
several rinses in TBS.
The primary antibodies used were rat anti-BrdU IgG (1 : 300,
Harlan, Loughborough, UK), mouse anti-BrdU IgG (1 : 400, Boehringer Mannheim), mouse antipolysialylated neural cell adhesion
molecule (PSA-NCAM) IgG (1 : 500, a generous gift of Dr T. Seki,
Juntendo University School of Medicine, Japan), mouse antiglial
brillary acidic protein (anti-GFAP) IgG (1 : 300, Chemicon, Temecula, USA), goat antidoublecortin IgG (1 : 400, Santa Cruz Biotechnology, Santa Cruz, CA, USA) goat anti-Sox-2 IgG (1 : 200, Santa
Cruz Biotechnology), rabbit anti-GFAP IgG (1 : 500, Dako, Glostrup,
Denmark) and rabbit anti-Ki67 IgG (1 : 100, Novocastra Laboratories,
Newcastle, UK). Secondary antibodies were uoroisothiocyanate
(FITC)-conjugated donkey antirat and antimouse IgG (1 : 150), Texas
Red-conjugated antigoat IgG (1 : 150), Cy5-conjugated donkey
antigoat and antirabbit IgG (1 : 150; all from Jackson Immunoresearch, West Grove, PA, USA) and biotinylated rat-adsorbed horse
antimouse IgG (1 : 125, Vector Laboratories, Kemila, Stockholm). No
unspecic labeling was observed when omitting incubation with
primary antibody.

All experimental and surgical protocols were approved by the Ethics

Committee for Animal Research at Lund University. Six-month-old
male spontaneously hypertensive rats (Mllegaard Breeding and
Research Center A S, Copenhagen, Denmark), weighing 300370 g,
were preoperatively housed in standard cages (550 350 200 mm,
34 rats per cage). The rats were anesthetized with methohexital
sodium, 50 mg kg i.p., and body temperature was kept close to
37
C. After a small craniotomy, the right middle cerebral artery
(MCA) was ligated distal to the origin of the striatal branches in 29
rats. In contrast to other rat strains, this procedure results in
spontaneously hypertensive rats in a consistent neocortical infarct
(Coyle, 1982; Grabowski et al., 1995). Whereas most studies use very
young rats at the age of 1012 weeks when they have just reached
sexual maturity according to veterinarians and breeders, the rats used
in our study were adult.
Postoperatively, the animals were all housed in individual cages for
about 24 h. Ten rats were then returned to standard cages, ten
transferred to enriched environment and nine placed in individual
cages equipped with a running wheel (diameter 350 mm, Techniplast
Gadazza, Buguggiate, Varese, Italy). Nine nonoperated rats were
placed in individual cages with running wheels and ten nonoperated
rats were housed in standard cages. Rats housed with running wheels
had not had any previous training in the wheel cages. The thymidine
analog BrdU (Boehringer Mannheim, Scandinavia AB, Bromma,
Sweden), which incorporates into the DNA of cells in the S-phase of
the cell cycle, was given as a daily i.p. injection starting 24 h after the
arterial ligation for 7 days (50 mg kg) and in the same way to
nonoperated rats. The enriched housing consisted of
815 610 1280-mm cages, equipped with horizontal and vertical
boards, chains, swings, wooden blocks, and objects of different sizes
and materials. The distance between the boards and the objects was
changed twice a week.

Behavioral testing
Preoperatively and 8 days after the ligation the coordination and
integration of movement was tested on a horizontal pole, rotating at 3
or 10 turns min. The pole, 45 mm in diameter and 1.5 m in length,
rotated alternately to the left or to the right, at 3 or 10 turns min.
Scores were assigned as follows: 0, the rat falls down; 1, the rat is
unable to traverse the pole but does not fall down; 2, the rat falls down
while attempting to cross the pole; 3, the rat jumps with both hind
limbs together, apparently supporting the weak hind limb with the
opposite strong limb; 4, the affected hind limb is used for less than
50% of the steps; 5, the rat crosses the pole with a few foot slips; 6, the
rat crosses the pole with no foot slips.

Tissue preparation
The day following the last BrdU injection the rats were deeply
anesthetized with an overdose of sodium pentobarbital and perfused
transcardially with saline solution followed by 4% paraformaldehyde
in 0.1 m phosphate buffer. Brains were removed, postxed overnight
in 4% paraformaldehyde in 0.1 m phosphate buffer and thereafter
transferred to 30% sucrose. Coronal sectioning (40 lm) was performed on a freezing microtome and sections were stored in tissue
cryoprotectant solution (25% ethylene glycol, 25% glycerol and 0.1 m
phosphate buffer) at )20
C.

Labeling of apoptotic cells

In order to assess apoptotic cell death, the terminal deoxynucleotidyl
transferase-mediated dUTP in situ nick-end labeling (TUNEL) method
was used according to the protocol supplied by the manufacturer
(Apoptag Peroxidase In situ apoptosis detection kit, Serologicals
Corp., AH Diagnostics, Sweden). Briey, sections were treated with
H2O2 to block endogenous peroxidase activity. Terminal deoxynucleotidyl transferase and digoxigenin-labeled nucleotides were added,
followed by incubation at 37
C for 1 h. After rinsing, peroxidaseconjugated antidigoxigenin IgG was added, the sections were
incubated for 30 min at room temperature and ultimately reacted with
the DAB-containing solution as described above.

Cell sampling and counting

Ki67-, BrdU- and TUNEL-positive cells were quantied in the SVZ of
each animal in ve coronal sections 240 lm apart. The SVZ was
dened as a 50-lm-wide band along the entire length of the lateral
wall of the forebrain lateral ventricle, between the crossing of the
corpus callosum and the crossing of the anterior commissure. Because
the current BrdU administration protocol spanned the 7 days before

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Postsichemic exercise, enrichment and cell genesis 2399

the animals were killed, the numbers of BrdU-positive cells in the
SVZ could not only reect effects on cell proliferation but also
possible effects on cell migration. At this early time point after the
cortical infarct neuronal recruitment to the infarct was not evident.
Newborn neurons migrate normally via the rostral migratory stream
(RMS) to the olfactory bulb to become new interneurons (Lois &
Alvarez-Buylla, 1994). Therefore, BrdU-positive cells were also
quantied in the RMS of each animal in ve sections 40 lm apart.
The RMS was dened as the area of doublecortin immunoreactivity
just medially of the anterior commissure at the level of the forceps
minor on coronal sections. TUNEL-positive cells in the SVZ were
counted through the entire thickness of the section. Because of the
large numbers of Ki67- and BrdU-immunopositive cells in the SVZ
and the RMS cell numbers were estimated semiquantitatively with the
following approach. Micrographs of stained sections were taken with a
20 objective lens and the area of specic staining along the entire
SVZ and in the RMS was measured with Scion Image software. Cell
numbers were then semiquantitatively calculated by dividing the area
of specic staining with the average area of an immunopositive cell.
In the dentate gyrus, counts of Ki67-positive cells were performed
in the SGZ whereas TUNEL- and BrdU-positive cells were counted
both in the SGZ and in the GCL, in order to include newborn cells that
had started to migrate into the GCL. The SGZ was dened as a 20-lmband immediately adjacent to the hilar surface of the GCL. Cells were
counted in ten anatomically matched coronal sections per animal,
240 lm apart.
Area measurements of the length of the lateral ventricle wall and the
area of GCL, respectively, were performed using an Intuos Graphics
tablet (Wacom, Japan) and digital image processing software (Nikon,
Goteborg, Sweden). A Nikon Diaphot Eclipse E600 microscope
connected to a video camera was used for cell counts and area
measurements. Semiquantitatively determined cell numbers in the
SVZ were expressed as cells per mm2 of SVZ area due to the fact that
image sampling only occurred in one focal plane, whereas manually
counted cell numbers were expressed as cells per mm3 of SVZ and
GCL, respectively (cross-sectional area 40 lm section thickness) as
the entire thickness of the section was sampled.

Phenotype determination of proliferating and newborn cells

A further aim was to distinguish between the dividing components of
the germinal niches: the GFAP-expressing astroglia, which are
putative neural stem cells, the intermediary transit amplifying neural
progenitor cells and the neuroblasts (Doetsch et al., 1997, 1999; Seri
et al., 2001; Alvarez-Buylla & Lim, 2004). To that end we used
multiple immunolabeling for the proliferation marker Ki67 (Kee et al.,
2002), GFAP, the transcription factor Sox-2, which is essentially
absent in immature neurons but is expressed by neural progenitors and
GFAP-immunoreactive astroglia (Bylund et al., 2003; Graham et al.,
2003; Komitova & Eriksson, 2004), and the immature neuronal
marker PSA-NCAM (Doetsch et al., 1997). Dividing putative neural
stem cells were identied as Ki67+ Sox-2+ GFAP+ triple labeled
cells, whereas dividing neural progenitors and neuronal precursors
were identied as Ki67+ Sox-2+ GFAP and Ki67+ PSA-NCAM+
double labeled cells, respectively. As Ki67+ Sox-2+ GFAP cells did
not appear to be a homogeneous population we distinguished between
two main groups: strongly Sox-2-positive cells (Sox-2Hi), which we
believe are early neural progenitors, and moderately to weakly Sox-2positive cells (Sox-2Lo), which we believe are late neural progenitors.
These assumptions are based on the fact that Sox-2 has been shown to
maintain neural progenitor identity during brain development and to

inhibit neuronal differentiation (Bylund et al., 2003; Graham et al.,

2003). Moreover, judging by our own observations in the adult
germinal niches Sox-2Hi cells were never found to colocalize with
immature neuronal markers, whereas weakly Sox-2-positive cells
could occasionally colabel for PSA-NCAM or doublecortin, indicating
that decreasing Sox-2 expression could be correlated with the
appearance of phenotypical characteristics of neuronal differentiation.
Colocalization of cell-specic markers was determined with
confocal laser scanning microscopy (Leica TCS SP2, Leica Microsystems, Heidelberg, Germany). At least 100 and 50 Ki67-labeled
cells per animal were characterized in the SVZ and the SGZ of the
dentate gyrus, respectively. Absolute numbers of Ki67-immunopositive cells expressing the different markers were calculated by
multiplying the corresponding fractions with the total numbers of
proliferating cells. The phenotype of BrdU-positive cells was examined in BrdU, doublecortin and GFAP triple stained sections of the
SVZ, RMS and the dentate gyrus of at least three animals per group.

Measurement of total tissue loss

Measurements of ipsi- and contralateral cortical cross-sectional area
down to the rhinal ssure were performed for each animal on ten
Nissl-stained sections 960 lm apart. The cortical volume was
calculated from the cross-sectional area and the distance between
the sections according to the Cavalieri principle (Gundersen et al.,
1988). The cortical infarct was expressed as percentage of the
contralateral cortical volume.

Statistical analysis
Behavioral scores are presented as median and 25% upper and lower
percentiles. KruskalWallis nonparametric analysis of variance with a
multiple comparison posthoc test at a signicance level of 95% was
used to determine differences between the groups in the behavioral
test. Statistical analysis was performed with one-way analysis of
variance (anova), followed by Tukey Kramer or Dunnetts posthoc
test for comparisons between the lesioned groups or between lesioned
groups and controls, respectively (Statview 4.01 for Macintosh).
Numerical data are presented as means SEM unless indicated
otherwise.

Results
There was no postoperative mortality or signicant weight changes in
any of the groups. Mean daily running distance for intact and lesioned
rats is shown in Fig. 1A.
Voluntary running activity gradually increased during the 7 days of
the experiment. A number of studies have shown that rats with free
exposure to running wheels run less the rst week, whether or not they
are lesioned, and that running distance gradually increases, subsequently
reaching a plateau (Shyu et al., 1984; Risedal et al., 2002; Makatsori
et al., 2003; Schwendt et al., 2003; Persson et al., 2004). Rats do not
have any apparent problems in running with the current model of cortical
infarct. However, lesioned rats ran on average less than intact rats.
Running distances were well above the threshold level of 500 m day
reported to be needed for up-regulation of some key gene products
believed to mediate effects of exercise on the brain (Shen et al., 2001).
One intact running rat ran much less than the others for 3 days and
then stopped running and was therefore not included in the study. One
operated rat in the standard environment was excluded because it did
not have any infarct.

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2400 M. Komitova et al.

Fig. 1. (A) Daily running distance of intact rats and rats with middle cerebral artery occlusion (MCAo) housed in individual cages with access to a running wheel.
Values presented are means SD (*P < 0.05). (B) Performance on a horizontal pole rotating at 3 and 10 turns min, respectively, preoperatively (preop) and 8 days
after MCAo for rats housed in standard environment, enriched environment or in individual cages with access to a running wheel. Behavioral scores are presented as
median and 25% upper and lower percentiles. The highest score, 6, indicates that the rats managed the tests without any problems (*P < 0.05 compared with
preoperative values and stroke enriched rats).

Total tissue loss

The volume of the cortical infarct did not differ signicantly between
the groups (stroke standard environment 49 2%, stroke enriched
environment 47 3%, stroke running wheel 51 1% of the volume
of contralateral cortex).

Behavioral testing
All the rats managed the rotating horizontal rod before the operation or
intervention (running) started. Neither group of intact rats (standard
environment or running wheel) changed their performance. In the
postischemic groups, lesioned enriched rats did not differ from intact
animals, whereas lesioned runners and lesioned rats in standard
environment performed signicantly worse than the lesioned enriched
rats, as well as compared with their own preoperative values (Fig. 1B).

Proliferating cells in the SVZ

The total numbers of Ki67-labeled cells in the SVZ were signicantly
increased bilaterally in all three lesioned groups compared with intact
animals housed in standard environment. However, Ki67-labeled cells

in the stroke running group were bilaterally lower than in the stroke
enriched group (Fig. 2A).

Apoptotic cells in the SVZ

The numbers of TUNEL-positive cells in the SVZ tended to be lower
in the lesioned groups than in the intact standard group but were found
to be signicantly decreased only in the ipsilateral SVZ in stroke
enriched rats (Fig. 2A). There were no signicant differences in the
numbers of TUNEL-positive cells between the lesioned groups.

Proliferating putative neural stem cells in the SVZ

Ki67+ Sox-2+ GFAP+ cells constituted 211% of the Ki67-positive
cells in the SVZ (Fig. 2B, left graph and photo montage). In the
contralateral SVZ, the fraction of Ki67+ Sox-2+ GFAP+ cells was
signicantly higher in the stroke enriched group (11 2%) than in the
other lesioned groups (stroke standard environment 2 1%, stroke
running wheel 3 0.4%, intact standard environment 3 1%).
Ipsilaterally, the absolute numbers of Ki67+ Sox-2+ GFAP+ triple
labeled cells were signicantly higher in the stroke standard and in the
stroke enriched groups than in intact standard and stroke runners,

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Postsichemic exercise, enrichment and cell genesis 2401

Fig. 2. (A) Quantication and representative micrographs of Ki67- and TUNEL-labeled cells (inset, apoptotic cell with typical morphology) in the SVZ.
(B) Phenotype determination of Ki67-positive cells in the SVZ and calculation of absolute cell numbers based on the fractions of colabeled cells. Ki67immunopositive cells were identied as putative neural stem cells (Ki67+ Sox-2+ GFAP+ triple labeled; left graph and photo montage), early highly Sox-2-positive
neural progenitors (Ki67+ Sox-2Hi GFAP double labeled; middle graph and photo montage, cells indicated by asterisks) and late moderate to low Sox-2-positive
neural progenitors (Ki67+ Sox-2Lo GFAP double labeled; middle graph and photo montage, most of the Ki67-positive cells not labeled by asterisks) as well as
neuroblasts (Ki67+ PSA-NCAM+ double labeled; left photo montage) which were mostly Sox-2 negative. (C) Quantication of BrdU-positive cells in the SVZ and
RMS. The majority of BrdU-positive cells in the SVZ and RMS, labeled with the current protocol of seven daily BrdU injections before the animals were killed,
expressed doublecortin (DCX). Values are presented as means SEM (*signicant difference compared to intact standard, signicant difference compared with
stroke runners, signicant difference compared with stroke standard, P < 0.05). Scale bars, 50 lm in A and C, 5 lm in B. IS, intact rats in standard environment;
IW, intact rats with access to a running wheel; SS, SE and SW, stroke-lesioned animals in standard environment, enriched environment and with access to a running
wheel postischemically, respectively.

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2402 M. Komitova et al.

which did not differ from intact animals. Contralaterally, the stroke
enriched group exhibited signicantly higher numbers of Ki67+ Sox2+ GFAP+ cells than the intact standard, stroke standard and stroke
runners group, the latter two being at the same level as intact animals.

Proliferating neural progenitor cells in the SVZ

Most of the Ki67-positive cells in the SVZ were Sox-2 immunopositive and GFAP immunonegative (
90%) (Fig. 2B, middle graph and
photo montage). Approximately one-quarter of Ki67+ Sox-2+ GFAP
cells were found to be Sox-2Hi. The percentage of Sox-2-positive cells
among proliferating cells was not inuenced by lesion or different
postischemic housing. The absolute numbers of Ki67, Sox-2Hi double
labeled cells were signicantly increased compared with intact
standard ipsilaterally in all three stroke groups. However, contralaterally, they were signicantly increased compared with intact standard
only in the stroke standard and in the stroke enriched animals but not
in stroke runners. The stroke standard and the stroke enriched groups
had signicantly more Ki67+ Sox-2Hi GFAP cells bilaterally than
did stroke runners. The absolute numbers of Ki67+ Sox-2Lo GFAP
cells were increased in all lesioned groups with a nonsignicant trend
towards higher numbers in stroke enriched compared with stroke
runners (P < 0.07).

SVZ cell genesis in intact runners

Cell proliferation and neurogenesis was thus found to be lower in the
SVZ of stroke runners. These effects were specic for the lesioninduced activation of the SVZ because basal cell genesis was not
signicantly altered in intact runners compared with intact nonrunners
housed in standard environment (Fig. 2A and C). The phenotype of
proliferating SVZ cells in intact running animals was therefore not
explored further.

Cell genesis and apoptosis in the SGZ of the dentate gyrus

The numbers of BrdU- and Ki67-labeled cells were bilaterally
increased in all stroke groups, on average > 5 times on the ipsilateral
and > 3 times on the contralateral side, without any signicant
differences resulting from postischemic intervention (Fig. 3A). There
were no signicant differences in the numbers of BrdU-positive cells

Proliferating neuroblasts in the SVZ

Around 3% of the Ki67-positive cells in the SVZ expressed PSANCAM and were thus identied as proliferating neuroblasts (Fig. 2B,
right graph and photo montage). The relative distribution of PSANCAM-labeled cells among proliferating cells did not differ signicantly between the groups. When the absolute numbers of Ki67+
PSA-NCAM+ cells were considered, stroke standard and stroke
enriched rats had signicantly higher numbers in the SVZ bilaterally
compared with stroke runners and intact standard, whereas the
numbers of proliferating neuroblasts in stroke runners did not differ
compared with those of the intact standard group. Moreover, stroke
enriched rats exhibited a further signicant increase in the number of
proliferating neuronal precursors in the contralateral SVZ compared
with stroke rats housed in standard environment postischemically.

Newborn BrdU-labeled cells in the SVZ and the RMS

The generation of new cells in the SVZ was also assessed by
quantifying the numbers of BrdU-labeled cells after seven daily
injections with BrdU. Stroke enriched animals displayed higher
numbers of BrdU-positive cells bilaterally than did stroke runners and
ipsilaterally than did intact standard, and lesioned rats in standard
environment had signicantly higher numbers of BrdU-labeled cells in
the contralateral SVZ compared with stroke runners, which in turn did
not differ signicantly from intact controls as regards the number of
BrdU-positive cells on either side (Fig. 2C). The majority of the BrdUlabeled, newborn cells in the SVZ were found to colabel with the
immature neuronal marker doublecortin (Fig. 2C), which labeled the
same cell populations as PSA-NCAM in the SVZ.
In the RMS, the numbers of BrdU-labeled cells, most of which
colabeled with doublecortin, tended to be lower in stroke runners with
a signicant difference in comparison with stroke standard ipsilaterally. A pattern similar to that observed in the SVZ suggests fewer
migrating neurons owing to decreased neurogenesis in the SVZ and
lack of effects of postischemic running on neuronal migration in the
forebrain (Fig. 2C).

Fig. 3. (A) Quantication of Ki67- and BrdU-labeled cells in the dentate

gyrus SGZ and GCL. (B) The majority of the Ki67-positive cells in the SGZ
were Sox-2 positive. Double arrows indicate a cluster of weekly Sox-2-positive
proliferating cells and the single arrow indicates a cluster of highly Sox-2positive proliferating cells. (B) Within the latter cell cluster there is a
Ki67+ Sox-2+ GFAP+ triple labeled radial glia-like cell that extends a GFAPpositive process (arrow) into the GCL. The majority of BrdU-positive cells in
the SGZ and GCL, labeled with the current protocol of seven daily BrdU
injections before the animals were killed, did not express Sox-2 (C) but
expressed the immature neuronal marker doublecortin (DCX) (D). Scale bars,
20 lm.

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Postsichemic exercise, enrichment and cell genesis 2403

between intact runners and nonrunners. There was, however, a trend
for an increase in the number of Ki67-positive cells in intact runners
vs. intact nonrunners (P 0.072).
TUNEL-positive cells were rarely found in the SGZ and the GCL of
intact controls and there were no signicant differences in apoptosis
rate due to lesion or different postischemic housing (data not shown).

Phenotype of the proliferating and newborn cells in SGZ

of the dentate gyrus
Around 8% of the proliferating cells were Ki67+ Sox-2+ GFAP+
triple labeled cells and one-quarter of these astroglial cells displayed
morphological features of radial glia-like cells, i.e. cell soma in the
SGZ and a radial process that traversed the GCL (Fig. 3B and B). The
majority of the proliferating cells in the SGZ were Ki67+ Sox-2+
GFAP (
66%) and approximately one-third of these proliferating
neural progenitor cells were found to be Sox-2Hi (Fig. 3B). Almost 5%
of the proliferating cells in the SGZ were PSA-NCAM-positive
immature neurons. Around 20% of the Ki67-positive cells did not
colabel for any of the above-mentioned markers and the majority of
these cells were present within the clusters of proliferating cells,
expressing the above-mentioned markers. There were no signicant
differences in the relative distribution of proliferating cell types among
the groups. The three stroke groups had thus bilaterally signicantly
increased absolute numbers of proliferating radial glia-like and
nonradial glia-like astroglia (Ki67+ Sox-2+ GFAP+), neural progenitors (Ki67+ Sox-2Hi,Lo GFAP) and immature neurons (Ki67+ PSANCAM+) as well as proliferating cells that did not colabel for any of
the above-mentioned markers (data not shown). There were, however,
no signicant effects of different postischemic housing on the different
subtypes of proliferating cells in the dentate gyrus. Intact runners
displayed signicantly increased numbers of proliferating nonradial
glia-like astroglia (Ki67+ Sox-2+ GFAP+) as well as neural progenitors (Ki67+ Sox-2Hi,Lo GFAP) in the SGZ compared with intact
controls housed in standard environment (data not shown).
The majority of BrdU-labeled newborn cells in the SGZ and the
GCL no longer expressed Sox-2 (Fig. 3C) and had assumed a neuronal
phenotype as reected by colabeling of BrdU with doublecortin
(Fig. 3D).

Discussion
This study shows that postischemic voluntary exercise and environmental enrichment modulated the lesion-induced increase in cell
proliferation in the SVZ but did not affect activation of the dentate
gyrus SGZ early after a cortical infarct.
Whereas running activity lacked effects on basal levels of SVZ cell
genesis in intact rats, in stroke animals it attenuated the lesion-induced
increase in proliferating neural stem progenitor cells and neuronal
precursors in the SVZ. Negative effects of exercise on lesion-induced
neural progenitor cell proliferation have been reported previously in
the dentate gyrus after transient global ischemia leading to hippocampal damage (Lee et al., 2003).
In contrast to the observations in wheel running lesioned animals,
there were no attenuating effects on lesion-induced SVZ cell genesis
in enriched rats and there was even an enhancement of the numbers
of proliferating putative neural stem cells and neuronal precursors
in the contralateral SVZ. There are indications that postischemic
environmental enrichment has more pronounced effects at a later point
in time when the lesion-induced proliferative stimulus has subsided
(M. Komitova et al., 2005).

Several studies have shown that the early phase after a brain lesion
is a vulnerable period. Data from different brain injury models indicate
that early initiation of physical activity, including voluntary exercise,
negatively inuences several parameters such as functional recovery,
lesion volume and the lesion-induced up-regulation of plasticityrelated proteins (Humm et al., 1998; Risedal et al., 1999; Griesbach
et al., 2004a,b). A number of studies have documented a benecial
effect of postischemic environmental enrichment on functional
outcome after focal brain ischemia and several comparative studies
have shown that postischemic wheel running does not have such
recovery-promoting effects, in agreement with gene expression data
(Johansson & Ohlsson, 1996; Risedal et al., 2002; Dahlqvist et al.,
2003; Johansson, 2004). In contrast to wheel running animals, which
are solely exposed to repetitive physical activity, enriched animals are
kept in a complex environment with opportunities for learning
experiences due to physical, sensory and social interactions with the
various components of the environment. Adaptive processes in the
brain initiated by the two treatments differ on the structural level.
Motor learning promotes synaptogenesis and induces plasticity of
cortical representation areas; in contrast, repetitive motor activity
induces angiogenesis, likely due to increased synaptic signaling and
metabolic demand (Black et al., 1990; Kleim et al., 1998; Plautz et al.,
2000). Moreover, housing in complex environmental conditions but
not treadmill running enhanced synaptic plasticity in the CA1 region
after global cerebral ischemia (Briones et al., 2004).
The functional signicance of lesion-induced activation of the SVZ
and modulation thereof remain to be established in future studies. The
current results indicate a functional improvement back to preoperative
values in postischemically enriched rats but not in lesioned exercising
rats or lesioned rats housed in standard environment. This early
functional improvement is most likely due to induction of other types of
plasticity than neuronal replacement, because newborn neurons would
have to be recruited, become mature and integrate into the circuitry in
order to contribute to functional recovery. A study utilizing the same
experimental stroke model demonstrated that postischemic environmental enrichment increased dendritic spine density in the homeotopic
contralateral cortex (Johansson & Belichenko, 2002). At the current
early time point after the cortical infarct neuronal recruitment to the
infarct itself was not evident, even though migratory PSA-NCAM- and
doublecortin-positive neuroblasts were occasionally encountered in the
ipsilateral corpus callosum. Robust recruitment of neuronal precursors
to the infarct is, however, evident at a later time point after a cortical
stroke (M. Komitova et al., 2005).
The mediators of the lesion-induced response of the adult germinal
niches have only recently begun to be elucidated. A number of growth
factors with known stimulatory effects on neural progenitor cells have
been shown to be induced by ischemia, suggesting their involvement
in the lesion-induced response of the SVZ and the dentate gyrus SGZ
(Kovacs et al., 1996; Jin et al., 2002; Kokaia & Lindvall, 2003). One
source of pro-regenerative peptide factors are astrocytes activated by
brain injury (Frautschy et al., 1991; Kovacs et al., 1996; Liberto et al.,
2004). Running activity has been shown to decrease the numbers of
astroglial cells and their activation after stroke (Ang et al., 2004) and
this might interfere with the stroke-induced increase in neural
progenitor cell proliferation. There are also indications in our material
that astrocytic activation might be decreased in stroke-lesioned
exercising rats (M.K. et al., unpublished observations).
Another possible mechanism through which postischemic exercise
might interfere with activation of the SVZ neural stem progenitor cell
compartment early after a cortical infarct is increased glutamatergic
signaling, which has been implicated in the exacerbation of brain
injury observed after early overuse of the impaired limb (Humm et al.,

2005 Federation of European Neuroscience Societies, European Journal of Neuroscience, 21, 23972405

2404 M. Komitova et al.

1999). Inhibitory effects of glutamate on neural progenitor cell
proliferation have been demonstrated in the embryonic SVZ (Haydar
et al., 2000). A focal ischemic lesion leads to increased glutamate
sensitivity in perilesional and contralateral cortical regions (Qu et al.,
1998) and exercise has been shown to increase glutamate levels in the
striatum (Meeusen et al., 1997). It is thus possible that early postlesion
exercise could cause excessive glutamatergic activation with subsequent negative effects on neural stem progenitor cell dynamics.
Postischemic environmental enrichment, by contrast, decreases levels
of brain-derived neurotrophic factor during the rst 2 weeks postlesion and has been hypothesized to decrease thereby hyperexcitability
after stroke (Zhao et al., 2000, 2001).
In conclusion, to the best of our knowledge this is the rst study that
has examined and compared the effects of environmental enrichment
and voluntary exercise on cell genesis in the adult germinal niches
early after an ischemic insult. We observed that postischemic exercise
and enriched environment inuenced the SVZ differentially but lacked
effects on the dentate gyrus SGZ. Whereas enriched environment had
certain enhancing effects on lesion-induced activation of the SVZ,
postischemic wheel running exerted attenuating effects and may thus
negatively inuence the contingent of new neuroblasts for potential
recruitment to the lesion.

Acknowledgements
This work was supported by grants from the Swedish Medical Research
Council, the Faculty of Medicine at Goteborg University, the Royal Physiographic Society in Lund, the Goteborg Society of Medicine, and the Fundraising Foundation for Neurological Research at the Department of Neurology
at Sahlgrenska University Hospital. Ms Pia Larsson is acknowledged for
assistance with area measurements.

Abbreviations
BrdU, bromodeoxyuridine; GCL, granule cell layer; GFAP, glial brillary
acidic protein; PSA-NCAM, polysialylated neural cell adhesion molecule;
RMS, rostral migratory stream; SGZ, subgranular zone of the dentate gyrus;
SVZ, subventricular zone; TUNEL, terminal deoxynucleotidyl transferasemediated dUTP in situ nick-end labeling.

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