n-methyl-d-aspartate receptor-mediated increase of neurogenesis in adult rat dentate gyrus following...

N-methyl-D-aspartate receptor-mediated increase ofneurogenesis in adult rat dentate gyrus following stroke

Andreas Arvidsson, Zaal Kokaia and Olle LindvallSection of Restorative Neurology, Wallenberg Neuroscience Center, BMC A11, University Hospital, SE-221 84, Lund, Sweden

Keywords: BrdU, Focal cerebral ischemia, glutamate, granule cells, neuronal proliferation

Abstract

Neurogenesis in the adult rat dentate gyrus was studied following focal ischemic insults produced by middle cerebral arteryocclusion (MCAO). Animals were subjected to either 30 min of MCAO, which causes damage con®ned to the striatum, or 2 h of

MCAO, which leads to both striatal and cortical infarction. When compared to sham-operated rats, MCAO-rats showed a marked

increase of the number of cells double-labelled for 5-bromo-2¢-deoxyuridine-5¢-monophosphate (BrdU; injected during 4±6 dayspostischemia) and neuronal-speci®c antigen (NeuN; a marker of postmitotic neurons) in the ipsilateral dentate granule cell layer

and subgranular zone at 5 weeks following the 2 h insult. Only a modest and variable increase of BrdU-labelled cells was found

after 30 min of MCAO. The enhanced neurogenesis was not dependent on cell death in the hippocampus, and its magnitude was

not correlated to the degree of cortical damage. Systemic administration of the N-methyl-D-aspartate (NMDA) receptor blockerdizocilpine maleate (MK-801) completely suppressed the elevated neurogenesis following 2 h of MCAO. Our ®ndings indicate that

stroke leads to increased neurogenesis in the adult rat dentate gyrus through glutamatergic mechanisms acting on NMDA

receptors. This modulatory effect may be mediated through changes in the levels of several growth factors, which occur afterstroke, and could in¯uence various regulatory steps of neurogenesis.

Introduction

The generation of new neurons continues throughout adulthood in

the dentate gyrus (DG) of both animals and humans (Altman &

Das, 1965; Kaplan & Hinds, 1977; Eriksson et al., 1998). In the

rat DG, neuronal progenitor cells reside and proliferate in the

subgranular zone (SGZ), develop a neuronal phenotype (Kaplan &

Hinds, 1977; Cameron et al., 1993), migrate into the granule cell

layer (GCL) (Altman & Bayer, 1990; Seki & Arai, 1993), adopt

morphological characteristics of granule cells (Kaplan & Hinds,

1977; Cameron et al., 1993), and extend axonal projections to the

CA3 region (Stan®eld & Trice, 1988; Markakis & Gage, 1999).

Neurogenesis in the DG is modulated by many factors, e.g.

ageing (Seki & Arai, 1995; Kuhn et al., 1996), physical activity

(van Praag et al., 1999), learning (Gould et al., 1999), enriched

environment (Kempermann et al., 1997), psychosocial stress

(Gould et al., 1997), adrenal steroids (Cameron & Gould,

1994), oestrogen (Tanapat et al., 1999), insulin-like growth factor

(AÊ berg et al. 2000), and glutamate receptor activation (Cameron

et al., 1995).

Several insults to the brain have been shown to promote DG

neurogenesis. This is observed after death of DG neurons caused

by adrenalectomy, or excitotoxic or mechanical lesions (Cameron

& Gould, 1994; Gould & Tanapat, 1997). Also, other insults

which may lead to neuronal death in the hippocampus, like global

forebrain ischemia (Liu et al., 1998; Bernabeu & Sharp, 2000;

Kee et al. 2001) and epileptic seizures (Bengzon et al., 1997;

Parent et al., 1997; Gray & Sundstrom, 1998; Parent et al., 1998)

stimulate neurogenesis in the DG. These ®ndings raise the

possibility that the insult-induced increase of neurogenesis helps

to replenish damaged neuronal circuits. However, there is as yet

no direct evidence that the degenerated neurons are actually

replaced by the new ones. Also, cell death does not seem to be a

necessary prerequisite for increased neurogenesis following brain

insults. Neurogenesis may be enhanced by seizure activity that

does not lead to any detectable neuronal death, like after

perforant path stimulation (Parent et al., 1997) and in a rat

model of electroconvulsive therapy (Madsen et al. 2000). This

indicates that insult-induced neurogenesis is not necessarily

triggered by neuronal death but is also regulated by other

mechanisms.

In the present study, we have analysed cell proliferation and

neurogenesis after two focal ischemic insults induced by middle

cerebral artery occlusion (MCAO). We used 30 min of MCAO,

which leads to damage that is con®ned to the striatum, and 2 h of

MCAO, which causes both striatal and cortical infarction. The

objectives were two-fold: ®rst, to determine whether focal cerebral

ischemia can lead to increased neurogenesis in the DG and, if this is

the case, to assess whether the magnitude of neurogenesis is related to

the severity of the insult; second, as our experiments showed that

stroke gives rise to increased neurogenesis, to explore the possibility

that glutamate acting through receptors of the NMDA or (a-amino-3-

hydroxy-5-methyl-4-isoxazole propionic acid (AMPA) types is

involved in the neurogenic response. Glutamatergic transmission

has been reported to modulate the rate of basal neurogenesis

(Cameron et al., 1995; Gould et al., 1997), and is probably involved

in mediating the increased expression of early genes and growth

factors, which occurs in the hippocampus following MCAO and

might affect various steps of neurogenesis.

Correspondence: Andreas Arvidsson, as above.E-mail: [email protected]

Received 14 February 2001, revised 11 April 2001, accepted 20 April 2001

European Journal of Neuroscience, Vol. 14, pp. 10±18, 2001 ã Federation of European Neuroscience Societies

Materials and methods

Animals and experimental design

Male Wistar rats (300±370 g body weight; MoÈllegaard's Breeding

Center, Copenhagen, Denmark) were fasted overnight and subjected

to either 30 min or 2 h of MCAO (see below), or sham surgery in

four consecutive experiments. In the ®rst experiment, the animals

received intraperitoneal injections of the thymidine analogue BrdU

(50 mg/kg body weight, Sigma-Aldrich, St Louis, MO, USA) twice

daily during days 4±6 (n = 4), 11±13 (n = 6), or 18±20 (n = 3) after

2 h of MCAO or sham surgery (n = 3). Because BrdU incorporation

in the DG was found to be highest during days 4±6, all groups were

given BrdU injections during this time interval in the subsequent

experiments. In the second experiment, rats were subjected to 30 min

of MCAO (n = 20) or sham surgery (n = 17). In the third experiment,

the rats were treated with the NMDA-receptor antagonist dizocilpine

maleate (MK-801; n = 7), the AMPA-receptor antagonist 2, 3-

dihydroxy-6-nitro-7-sulfamoylbenzo(F)-quinoxaline (NBQX; n = 6),

or vehicle (n = 7) prior to 30 min of MCAO. Three groups of sham-

operated animals (n = 5 for each group) were given the same doses of

MK-801 or NBQX, or vehicle. In the fourth experiment, animals

received either vehicle or MK-801 prior to 2 h of MCAO, whereas all

sham-operated animals received vehicle injections (n = 10 for all

groups). To be able to determine if newborn cells expressed neuronal

markers, in all experiments the animals were allowed to survive for

28 days following the last BrdU injection.

If body temperature exceeded 39.5 °C at any time during, or in the

hours after, MCAO, or if a subarachnoid haemorrhage was found

upon inspection of the brain after perfusion, the animal was excluded.

In the second, third, and fourth experiments, all animals showing any

damage to the CA1 region of the hippocampus, as determined by

microscopy of sections immunohistochemically stained for the

neuronal marker NeuN, were also excluded.

Animal procedures were approved by the Research Ethical

Committee at the Medical Faculty of the University of Lund.

Induction of focal ischemia and drug administration

Transient MCAO was induced using the intraluminal ®lament

technique (Koizumi et al., 1986; Zhao et al., 1994; Kokaia et al.,

1995). Brie¯y, the rats were anaesthetized with halothane, intubated,

and arti®cially ventilated. Following isolation of the right carotid

arteries, the common and external carotids were proximally ligated

and the internal carotid was temporarily occluded with a micro-

vascular clip. Via an incision in the common carotid artery, a silicon

rubber (Silastic E RTV, Sikema, Stockholm, Sweden) coated nylon

mono®lament (0.25 mm diameter) with a rounded tip was then

advanced through the internal carotid artery until it presumably had

passed and occluded the origin of the middle cerebral artery. The rats

were then extubated and allowed to recover from anaesthesia. Thirty

min or 2 h after occlusion, the rats were brie¯y re-anaesthetized and

the occluding ®lament was withdrawn. Animals subjected to 2 h of

MCAO were cooled in a cold air ¯ow to avoid hyperthermia (Zhao

et al., 1994) whenever necessary. Sham surgeries were performed in

the same way, except that the ®lament was advanced only a few mm

inside the internal carotid artery. When glutamate receptor antag-

onists were given, MK-801 (Tocris, Bristol, UK), dissolved in saline,

was administered as a single intraperitoneal injection (3 mg/kg body

weight) at 15 min before MCAO, and NBQX (Novo-Nordisk,

Copenhagen, Denmark), dissolved in 100% dimethylsulfoxide

(DMSO), was given as three intraperitoneal doses (30 mg/kg each)

at 30, 20, and 5 min before occlusion. In the third experiment, vehicle

(DMSO) was given as three injections following the same time-

schedule as the NBQX injections, to all animals that were not given

NBQX. In the fourth experiment, vehicle (saline) was given 15 min

prior to MCAO. Arterial blood pressure and body temperature were

monitored during surgery, and ventilation was adjusted according to

arterial pO2, pCO2 and pH. Physiological parameters measured

immediately prior to MCAO and reperfusion are presented in

Table 1.

Immunohistochemistry

After transcardial perfusion of the rats with 4% ice-cold phosphate

buffered paraformaldehyde (PFA), the brains were post®xed in 4%

PFA and sectioned in the coronal plane at 20 and 40 mm, on

powdered dry ice, using a microtome. The sections were then double

stained for BrdU and NeuN. Brie¯y, free-¯oating sections were

denatured by incubation in 1 M hydrochloric acid at 65 °C for

TABLE 1. Physiological parameters in rats subjected to sham surgery or MCAO

Parameter

Experimental group

Sham 30 min MCAO 2 h MCAO 2 h MCAO + MK-801

At onset of occlusion:Blood pressure (mmHg) 108.00 6 19.00 96.00 6 18.00 91.00 6 13.00 140.00 6 19.00*²#

Arterial pO2 (mmHg) 111.40 6 10.10 108.50 6 10.10 110.00 6 12.60 102.80 6 7.50Arterial pCO2 (mmHg) 36.70 6 2.40 37.70 6 2.50 37.40 6 1.90 38.70 6 3.60Arterial pH 7.40 6 0.02 7.40 6 0.01 7.40 6 0.02 7.39 6 0.02Blood glucose (mM) 6.70 6 1.30 6.50 6 1.00 6.60 6 0.70 6.80 6 0.60Body temperature (°C) 37.50 6 0.30 37.60 6 0.30 37.70 6 0.30 37.80 6 0.40

At onset of reperfusion:Blood pressure (mmHg) 112.00 6 14.00 117.00 6 21.00 113.00 6 15.00 140.00 6 12.00*²#

Arterial pO2 (mmHg) 111.40 6 13.90 111.50 6 13.40 135.20 6 18.20²# 132.80 6 22.10²#

Arterial pCO2 (mmHg) 41.70 6 4.40 44.30 6 3.40 36.70 6 4.50*² 42.40 6 2.70#

Arterial pH 7.37 6 0.05 7.36 6 0.04 7.39 6 0.04 7.33 6 0.02#

Body temperature (°C) 37.40 6 0.60 37.90 6 0.50 38.30 6 0.60* 37.90 6 0.70

Values are expressed as means 6 SD. Measurements were made 1±10 min before insertion of the ®lament and 1±3 min before its withdrawal. `Sham' comprisesvehicle-injected, sham-operated animals from the second, third, and fourth experiments. `30 min MCAO' includes vehicle-injected animals subjected to 30 min ofMCAO (second and third experiments). `2 h MCAO' and `2 h MCAO + MK-801' consist of animals from the fourth experiment that were subjected to 2 h ofMCAO and treated with vehicle or MK-801, respectively. *different from `Sham'; ²different from `30 min MCAO', #different from `2 h MCAO', one-way ANOVA

followed by Bonferroni±Dunn post hoc test.

Neurogenesis after stroke 11

ã 2001 Federation of European Neuroscience Societies, European Journal of Neuroscience, 14, 10±18

30 min. Following rinsing, sections were preincubated for 1 h in a

mixture of 5% normal donkey serum (NDS) and 5% normal horse

serum (NHS) in phosphate buffered saline (PBS), after which they

were incubated for 36 h with 1 : 100 rat anti-BrdU antibody (Harlan

Sera-Lab, Loughborough, UK) and 1 : 100 mouse anti-NeuN anti-

body (Chemicon, Temecula, CA, USA) in preincubation solution.

The sections were then rinsed and incubated for 2 h with 1 : 200

secondary Cy3-conjugated donkey anti-rat antibody (Jackson

ImmunoResearch, West Grove, PA, USA) and 1 : 200 secondary

biotinylated horse anti-mouse antibody (Vector, Burlingame, CA,

USA) in a mixture of 2% NDS and 2% NHS in PBS. After a new

rinse, sections were incubated for 2 h with 1 : 200 Alexa 488-

conjugated avidin (Molecular Probes, Eugene, OR, USA) in PBS,

rinsed, mounted on super plus slides, and cover-slipped with PVA-

DABCO mounting medium.

Fluoro-Jade staining

Sections were washed several times in KPBS, mounted on glass

slides and dried. Then slides were immersed in absolute ethanol,

followed by 70% ethanol and distilled water. After incubation in

0.06% solution of potassium permanganate for 15 min, the slides

were rinsed in distilled water and transferred to the Fluoro-Jade

solution containing 0.01% Fluoro-Jade (Histo-Chem Inc., Jefferson,

AR, USA) and 0.1% acetic acid (1 : 9), placed on shaker for 30 min,

and after several washes in distilled water, were dried, immersed in

xylene and coverslipped.

Microscopical analysis

All counting of BrdU-immunopositive cells was performed at 40 3magni®cation on an epi¯uorescence microscope by an investigator

blind to treatment history. Labelled cells located within the NeuN-

positive dentate GCL or in the SGZ, de®ned as the area with the

height of a single dentate granule cell below the lowest unbroken row

of cells in the GCL, were counted. In the ®rst experiment, counting of

BrdU-positive, as well as cells double-labelled with BrdU and NeuN,

was performed using epi¯uorescence microscopy in three 40 mm

coronal sections spaced apart by 360 mm, starting at ±3.3 mm from

bregma and moving caudally. The numbers of BrdU- and BrdU/

NeuN-positive cells are presented as the sums of bilateral counts in

these three sections. In the other three experiments, counting of

BrdU-positive cells was performed in ®ve 40 mm coronal sections

spaced apart by 300 mm, starting at the same level as in the ®rst

experiment. The numbers of BrdU-positive cells in these experiments

are given as the sums of counts, separate for the side ipsilateral and

contralateral to MCAO, in these ®ve sections.

Analysis of the proportion of BrdU-positive cells coexpressing

NeuN in the fourth experiment was achieved using a Bio-Rad

confocal laser scanning microscope (Bio-Rad Microscience, Hemel

Hempstead, UK), by an investigator blind to treatment history, in

20 mm sections from all animals that were included in the analysis of

cell proliferation. For each animal, all BrdU-positive cells in the DG

ipsilateral to MCAO in at least two sections (minimum 50 cells, or all

BrdU-positive cells in the ipsilateral DG in 12 sections throughout the

hippocampus) were analysed. Cells were considered double-labelled

when BrdU and NeuN immunoreactivity was colocalized in a

minimum of three consecutive steps in a z-series (z-step 1 mm, iris

3.0) taken at 40 3 magni®cation.

Analysis of neuronal damage in the striatum and cerebral cortex

was performed in sections immunohistochemically stained for NeuN

using a semiquantitative scale: 0, no neuronal damage; 1, selective

neuronal loss; 2, patchy neuronal loss or small infarction; 3, large

infarction. In addition, the number of NeuN-positive cells in the

entorhinal cortex was counted bilaterally in one 20 mm coronal

section (±5.8 to ±6.0 mm from bregma) from each animal subjected

to 2 h of MCAO in the fourth experiment. Neuronal loss in the

entorhinal cortex ipsilateral to MCAO is expressed as the percentage

of lost neurons relative to the number of neurons on the contralateral

side.

Statistical analysis

Physiological parameters are presented as means 6 standard devi-

ation (SD) whereas other data are given as means 6 standard error of

the mean (SEM). Differences in physiological parameters were

analysed by ANOVA followed by Bonferroni±Dunn post hoc test.

Differences in counts of BrdU-immunopositive cells were evaluated

with unpaired t-test (second experiment) or one-way ANOVA followed

by Bonferroni±Dunn post hoc test (fourth experiment). Side differ-

ences in counts of BrdU-immunopositive cells in the DG or of NeuN-

positive cells in the entorhinal cortex were evaluated with a paired t-

test. Differences in neuronal damage scores were analysed with the

Chi squared test. Spearman's rank correlation coef®cient was used to

assess correlation between cortical damage scores, or neuronal loss in

the entorhinal cortex, and numbers of BrdU-positive cells in the DG.

Results

Physiological parameters and distribution of ischemic damage

Physiological parameters are summarized in Table 1. A slight

respiratory acidosis was seen at the onset of reperfusion in most

animals treated with MK-801 or NBQX (not shown). MK-801

administration also caused an increase in arterial blood pressure both

at the time of occlusion and reperfusion.

Ischemic damage was evaluated using immunohistochemistry for

the neuron-speci®c marker NeuN. In vehicle-treated animals, 2 h of

MCAO caused almost complete loss of neurons in all but the most

medial parts of the striatum and severe loss of neurons in the parietal

cortex. In contrast, MK-801-treated animals showed no, or only

selective, neuronal death in the cerebral cortex after 2 h of MCAO,

whereas the striatal damage was unchanged. Counting of NeuN-

positive cells revealed no signi®cant loss of neurons in the ipsilateral

entorhinal cortex in the vehicle- or MK-801-treated groups, although

moderate loss (47% and 33% of the neurons compared to the opposite

side) was seen in two animals of the vehicle-treated group. In

addition, to explore whether there was selective neuronal loss in the

entorhinal cortex, not detectable with NeuN staining, sections from

three animals from the vehicle-treated group and three animals from

the MK-801-treated group, that did not have decreased numbers of

NeuN-positive cells, were stained with Fluoro-Jade. This marker

stains degenerated neurons (Schmued et al., 1997) which are still

detectable several weeks after the insult (Larsson, E., Lindvall, O. &

Kokia, Z., unpublished observation). Microscopic examination of

sections did not reveal any Fluoro-Jade-positive cells in the

entorhinal cortex of these animals, although many stained cells

were clearly detectable in the ischemic parietal cortex (data not

shown).

Following 30 min of MCAO, the lesion was usually restricted to

the striatum, and similar in groups treated with vehicle, MK-801, or

NBQX. Four animals in the ®rst experiment, four animals in the

second experiment, one animal in the third experiment, and three

animals in the fourth experiment showed neuronal damage in the

CA1 ipsilateral to MCAO, with a pattern similar to that seen

bilaterally after global forebrain ischemia. In the second, third, and

12 A. Arvidsson et al.


fourth experiments, these animals were excluded from further

analysis.

Cell proliferation and neurogenesis following MCAO

In the ®rst experiment, we wanted to obtain evidence whether

increased neurogenesis could be detected after a focal ischemic insult

and, if this was the case, to determine the time interval of maximum

cell proliferation. As assessed at 28 days after the last BrdU injection,

the highest number of labelled cells (432% of sham) bilaterally in the

dentate GCL and SGZ was obtained when BrdU was injected at 4±

6 days after 2 h of MCAO (Fig. 1). Similarly, the maximum number

of BrdU-positive neurons double-stained with NeuN (370% of sham)

was detected after injection in this time interval. In contrast, no

differences were obtained when BrdU was injected at later time

intervals. In the subsequent experiments, we therefore injected BrdU

at 4±6 days after the focal ischemic insult, when the peak of neuronal

precursor proliferation seemed to occur.

In the second experiment, we explored the possibility that a less

severe insult, 30 min of MCAO, could give rise to increased cell

proliferation in the DG. Large, between animal variations were

observed, and no signi®cant increase in the numbers of BrdU-labelled

cells could be seen when counts ipsi- and contralateral to MCAO

were added together. The mean value was 135% of sham controls.

However, there was a signi®cantly increased number of BrdU-

labelled cells (to 194% of sham) on the side ipsilateral to the ischemic

insult when compared to sham-operated animals (Fig. 2). Whereas

there was no difference between the two sides in the sham group, the

number of BrdU-labelled cells was signi®cantly higher in the

ipsilateral when compared to the contralateral DG in the ischemic

rats. This provides evidence for the stimulation of cell proliferation

after 30 min of MCAO, albeit of lesser magnitude than following the

2 h insult.

In the third experiment we investigated whether the ischemia-

induced cell proliferation could be in¯uenced by blockade of NMDA

or AMPA glutamate receptors. Vehicle, MK-801, or NBQX was

administered to rats subjected to 30 min of MCAO. Whereas MK-

801 completely prevented the increase in the number of BrdU-

labelled cells, NBQX had no effect (Fig. 3). Similar injections of

MK-801 or NBQX in sham-operated animals did not alter the basal

rate of proliferation during the same time interval (total numbers of

BrdU-immunopositive cells in the ipsilateral DG in ®ve sections were

50 6 2 in vehicle injected animals, 69 6 22 cells in MK-801-treated

animals, and 51 6 16 cells in NBQX-treated animals,

means 6 SEM). Our ®ndings thus indicated that NMDA receptors

may mediate the effect of focal ischemia on cell proliferation,

whereas AMPA receptors seem to be less involved.

In the fourth experiment, we characterized the cell proliferation

and the effect of NMDA receptor blockade in more detail following

2 h of MCAO. As illustrated in Figs 4 and 5, the ischemic insult gave

rise to a marked increase (to 280% of sham) of the number of BrdU-

labelled cells in the dentate GCL and SGZ ipsilateral to the MCAO.

Although there seemed to be an increased number of BrdU-labelled

cells also on the contralateral side, they were considerably fewer

when compared to those in the ipsilateral DG. Administration of MK-

801 completely prevented the increase of the number of BrdU-

positive cells following the 2 h insult (Fig. 4). The proportion of

BrdU-positive cells double-labelled with NeuN in the ipsilateral DG

was similar in vehicle- and MK-801-treated animals subjected to 2 h

of MCAO (69 6 6% and 54 6 9%, respectively) and in animals

subjected to sham surgery (60 6 6%).

Because NMDA receptor blockade can also counteract ischemic

neuronal death (Park et al., 1988), we evaluated the degree of damage

to the cerebral cortex in vehicle- and MK-801-treated rats subjected

to 2 h of MCAO. Treatment with MK-801 led to signi®cantly less

damage to the cerebral cortex following the insult (Fig. 6A). We

therefore speculated that the inhibitory effect of MK-801 on cell

proliferation could be indirect and related to its neuroprotective

action. However, arguing against this hypothesis, the magnitude of

cell proliferation in the DG after 2 h of MCAO was not correlated to

the degree of cortical damage (Fig. 6B) or to the presence of neuronal

loss in the entorhinal cortex. Furthermore, no animal in any of the

groups showed ischemic damage in the hippocampus.

FIG. 2. Magnitude of cell proliferation in the DG after 30 min of MCAO orsham surgery. The bars show the total number of BrdU-immunoreactivecells in the granule cell layer and subgranular zone on the side ipsilateraland contralateral to ischemia in ®ve consecutive 40 mm sections. Datashown as means 6 SEM. *P < 0.05 compared to sham surgery, unpaired t-test. ²P < 0.05 for difference between sides, paired t-test. (n = 15 for shamgroup, n = 18 for MCAO group).

FIG. 1. Time course of cell proliferation and neurogenesis in the rat dentategyrus (DG) after 2 h of MCAO. The bars show the total number of BrdU-immunoreactive cells in the granule cell layer and subgranular zone on bothsides in three consecutive 40 mm sections. The hatched portions of the barsdepict the subpopulation of cells, which was also labelled with the neuron-speci®c marker NeuN, as determined with epi¯uorescence microscopy. Datashown as means 6 SEM. (n = 3, 4, 6, and 3 for sham, day 4±6, day 11±13,and day 18±20, respectively).



Discussion

This study shows that focal ischemic insults induced by MCAO lead

to increased neurogenesis in the ipsilateral DG. Whereas the increase

was modest and variable after 30 min of MCAO, the 2 h insult gave

rise to marked and robust enhancement of neurogenesis. The

increased neurogenesis was not dependent on the occurrence of cell

death in the hippocampus, and its magnitude was not correlated to the

degree of cortical damage. The NMDA receptor blocker MK-801

completely suppressed the increased neurogenesis following 2 h of

MCAO.

The highest number of BrdU-labelled cells, as well as of BrdU- and

NeuN-double-labelled cells, was detected when BrdU had been

administered 4±6 days after MCAO. When BrdU was given 11±

13 days or 18±20 days following the insult, no or only modest

changes of the number of labelled cells were observed. This

neurogenic response is, thus, similar to that shown after seizures

(Parent et al., 1997; Madsen et al. 2000) and global forebrain

ischemia (Liu et al., 1998; Kee et al. 2001), in that it is temporary,

results in a several-fold increase in the production of new neurons,

and occurs several days after the insult.

It may be argued that BrdU had been incorporated not in newly

divided cells, but in neurons, which were repairing damaged DNA

following the focal ischemia. However, our ®ndings strongly support

the interpretation that the increased number of BrdU-immunoreactive

and NeuN-positive cells in the DG was due to stimulation of

neurogenesis by the insult. First, we observed the BrdU-labelled cells

in the SGZ and GCL where neurogenesis has previously been

demonstrated (Gage, 2000). Second, no damage was observed in the

CA1 region or in the dentate GCL in the animals included in the

analysis. The CA1 pyramidal cells are much more vulnerable to

ischemia when compared to the dentate granule cells and it therefore

seems unlikely that these latter cells would be at all damaged. Third,

as discussed by Liu et al. (1998), cells with DNA strand breaks most

probably do not incorporate enough BrdU to be detected by

immunohistochemistry. Fourth, the BrdU-labelled cells survived for

more than 4 weeks after the insult and expressed the neuronal marker

NeuN, which is less likely to occur with severely damaged cells.

What are the mechanisms underlying the increased neurogenesis

after stroke and how does NMDA-receptor blockade interfere with

these mechanisms? Notably, the enhanced neurogenesis was observed

predominantly ipsilateral to the MCAO. In the present MCAO model,

hippocampus is not normally included in the densely ischemic area

(Memezawa et al., 1992a). Sixty minutes of MCAO does not change

local hippocampal blood ¯ow (Memezawa et al., 1992a), but after

15 min of recirculation there is a 58% reduction ipsilateral to the

insult (Memezawa et al., 1992b). Damage to the CA1 region,

indicative of hippocampal ischemia, may occur in some animals after

MCAO (Zhu & Auer, 1995), and was observed also here in about

10% of the rats. These animals were excluded from further analysis

and, thus, it is unlikely that the increased neurogenesis was caused by

ischemic damage to the hippocampus. Similarly, seizure activity

which is not associated with neuronal death can promote neurogen-

esis (Madsen et al. 2000). It is also unlikely that mild hippocampal

ischemia (that does not cause neuronal death) plays a major role in

the increased neurogenesis because the shortest duration of global

ischemia promoting cell proliferation in the gerbil DG is 4 min,

which is associated with damage to the CA1 and hilus (Liu et al.,

1998).

In a parallel study on gerbils (Bernabeu & Sharp, 2000), both the

massive CA1 neuronal death and the increased neurogenesis follow-

ing 10 min of global forebrain ischemia were blocked by systemic or

intrahippocampal injection of MK-801 or NBQX. The authors

proposed that in their model, decreased glutamate receptor activation,

secondary to chronic hippocampal injury, triggered neurogenesis. The

lack of effect of NBQX and the absence of detectable neuronal death

in our experiment suggest that the mechanisms modulating

FIG. 3. Effect of glutamate receptor blockade on the magnitude of cellproliferation in the DG following 30 min of MCAO. Bars show totalnumber of BrdU-immunoreactive cells in the granule cell layer andsubgranular zone on the side ipsilateral and contralateral to ischemia in ®veconsecutive 40 mm sections in animals subjected to 30 min of MCAO andtreated with vehicle, the NMDA receptor antagonist MK-801, or the AMPAreceptor antagonist NBQX. Data shown as means 6 SEM. (n = 6 forvehicle group, n = 7 for MK-801 group, n = 6 for NBQX group).

FIG. 4. Magnitude of cell proliferation and neurogenesis in the DG after 2 hof MCAO and effect of NMDA-receptor blockade. Bars show total numberof BrdU immunoreactive cells in the granule cell layer and subgranularzone on the side ipsilateral or contralateral to ischemia in ®ve consecutive40 mm sections in animals subjected to sham surgery, 2 h of MCAO withvehicle treatment, or 2 h of MCAO with MK-801 treatment. The hatchedportions of the bars depict the subpopulation of cells on the ipsilateral side,which was also labelled with NeuN, as determined using confocalmicroscopy. Data shown as means 6 SEM. *P < 0.05 compared to shamsurgery or 2 h of MCAO with MK-801 treatment, one-way ANOVA followedby Bonferroni±Dunn post hoc test. ²P < 0.05 for difference between sides,paired t-test. (n = 10 for sham-vehicle group, n = 9 for MCAO-vehiclegroup, n = 8 for MCAO-MK-801 group).



neurogenesis are at least partly different following MCAO and global

forebrain ischemia. One possibility could be that infarction in the

entorhinal cortex, which has been reported to occur following 2±3 h

of MCAO (Memezawa et al., 1992b), promotes neurogenesis. Thus,

excitotoxic lesions of the medial entorhinal cortex have been reported

to increase dentate neurogenesis (Cameron et al., 1995). However, we

observed neuronal loss in the entorhinal cortex only rarely after either

30 min or 2 h of MCAO. Furthermore, there was no correlation

FIG. 5. (A±D). Epi¯uorescence photomicrographs of immunoreactivity for BrdU (in red) and NeuN (in green) (A and B) or BrdU only (C and D) in the DGof 40 mm sections ipsilateral to sham surgery (A and C) or 2 h of MCAO (B and D). (E±J). Confocal microscopy images of immunoreactivity for BrdU (Eand H), NeuN (F and I) and both (G and J) from the same section as in B and D. E±G show the boxed region in the tip of the DG in D and H±I show theboxed region in the upper limb. Arrows depict some BrdU immunopositive cells that coexpress NeuN. All BrdU-positive cells in E±G are double labelled.Arrowhead in H±J shows a BrdU-labelled cell that does not express NeuN. Scale bar in J is 200 mm for A±D and 50 mm for E±J.



between the extent of damage to the entorhinal cortex and the

numbers of BrdU-positive cells in the ipsilateral DG after 2 h of

MCAO, which argues against damage to this cortical region as the

major mechanism promoting neurogenesis.

Hypothetically, the cascade of changes in the levels of various

growth factors in the hippocampal formation, which occurs following

MCAO, could be responsible for the enhanced neurogenesis. For

example, 1±2 h of transient MCAO leads to a long-lasting increase in

the synthesis of basic ®broblast growth factor (bFGF) mRNA (Lin

et al., 1997) unilaterally, and of brain-derived neurotrophic factor

(BDNF) mRNA (Kokaia et al., 1995) bilaterally, in the hippocampus.

Basic ®broblast growth factor is a mitogen for hippocampal

progenitor cells in vitro (Gage et al., 1995). Subcutaneous injections

of bFGF stimulate cell proliferation in the hippocampus of neonatal

and adolescent rats (Wagner et al., 1999), whereas no change has

been observed after either subcutaneous injections (Wagner et al.,

1999) or chronic intraventricular infusion (Kuhn et al., 1997) in adult

animals. This may be due to insuf®cient diffusion of bFGF to the

progenitor cells in the SGZ. BDNF administered intraventricularly

can increase the generation and/or survival of new neurons in the

adult rat olfactory bulb (Zigova et al., 1998). Changes in the synthesis

of several growth factors can be regulated by NMDA-receptor

mediated glutamatergic mechanisms. Glutamate increases the syn-

thesis of, e.g. nerve growth factor (NGF), BDNF, and bFGF mRNA

in vitro and this effect can be blocked by MK-801 (Zafra et al., 1991;

Uchida et al., 1998). Furthermore, electrical stimulation of the

perforant path, i.e. the main glutamatergic input to the DG,

originating in the entorhinal cortex, leads to ipsilateral up-regulation

of BDNF and NGF mRNA levels via NMDA receptors (Springer

et al., 1994), as well as increased cell proliferation (Parent et al.,

1997), in the DG. Focal cerebral ischemia is known to cause

spontaneous depolarization waves (Nedergaard & Astrup, 1986), that

can spread over large distances in the ipsilateral cortex (Hasegawa

et al., 1995; Dijkhuizen et al., 1999), most likely including also the

entorhinal cortex. In a hypothetical scenario, subsequent activation of

the glutamatergic input to the hippocampus, which is supported by

the ®ndings of increased hippocampal glutamate levels during

MCAO in the gerbil (Miyashita et al., 1994), leads to the changes

of growth factor synthesis and the increased neurogenesis observed

after this insult. The NMDA-receptor antagonist may inhibit

neurogenesis by blocking glutamatergic mechanisms at the entorhinal

or hippocampal level.

Three previous studies have indicated that MK-801 stimulates

basal neurogenesis in the normal adult brain. Administration of MK-

801 increased the density of 3H-thymidine-labelled cells or the

number of BrdU-labelled cells in the DG of Sprague±Dawley rats

(Cameron et al., 1995) or tree shrews (Gould et al., 1997)

respectively. The majority of BrdU-labelled cells were double-

stained with a neuronal marker. The results of Cameron et al. (1995)

and Gould et al. (1997) indicated that neurogenesis in the intact DG is

down-regulated by NMDA receptor activation. In these studies,

stimulation of neurogenesis was observed within hours after MK-801

treatment, indicating a direct and rapid modulatory effect by the

excitatory input in the normal brain. Recent evidence from Bernabeu

& Sharp (2000) suggest that this is not a short-term effect but that at

least in gerbils MK-801 administration during a limited time period

may alter basal cell proliferation for several days. We could not

reproduce this effect, using BrdU injections at 4±6 days following a

single MK-801 injection in sham-operated rats. This could be due to

that Bernabeu & Sharp (2000) used multiple doses of MK-801, and

that the animals were allowed to survive for only 3 days after the last

BrdU injection, compared to 28 days in our experiment. The most

likely explanation for the discrepancy between the effect of MK-801

on basal and MCAO-induced proliferation seems to be different

modes of action of NMDA-receptor mediated glutamatergic mech-

anisms in these processes. We propose that following stroke, the

marked stimulation of neurogenesis by glutamatergic mechanisms,

presumably mediated at least partly through alterations of growth

factor levels, overrides the more subtle, direct suppressant action of

these mechanisms observed under basal conditions.

In conclusion, the present ®ndings indicate that stroke leads to

increased neurogenesis in the adult rat DG through a mechanism

involving NMDA receptors. It is well established that release of

glutamate, acting on NMDA receptors, plays an important role for

ischemia-induced neuronal death (see Dirnagl et al., 1999).

Administration of an NMDA receptor blocker, e.g. MK-801 attenu-

ates the extent of tissue damage following focal ischemia (Park et al.,

1988). MCAO leads to impairment in tasks of spatial learning,

generally associated with hippocampal function, like the Morris

FIG. 6. (A) Effect of MK-801 treatment on neuronal damage in the cerebralcortex. Individual values (one dot for each animal) of semiquantitativeestimation of cortical damage in brains stained with NeuN immuno-histochemistry from animals treated with vehicle or MK-801 after 2 h ofMCAO. Scores: 0, no neuronal damage; 1, selective neuronal death; 2,patchy necrosis or small infarction and 3, large infarction. (B) Relationbetween cortical damage score (horizontal-axis) and total number of BrdU-immunoreactive cells in the dentate gyrus granule cell layer and subgranularzone ipsilateral to ischemia in ®ve consecutive 40 mm sections (vertical-axis) after 2 h of MCAO. Values from animals treated with vehicle (closedsquares) or MK-801 (open circles) are plotted individually. The linerepresents regression for all data. There is no signi®cant correlation betweencortical damage and proliferation (P > 0.05, Spearmans r = 0.23). *P < 0.05compared to vehicle treatment, Chi squared test.



water-maze (Markgraf et al., 1992; Yonemori et al., 1999). If the

increased neurogenesis demonstrated here is a neuroregenerative

response which aims at restoring or preserving hippocampal function

following stroke, treatment with NMDA-receptor antagonists may in

fact also have negative effects on the degree of recovery. On the other

hand, if increased neurogenesis has pathological consequences, e.g.

by enhancing the susceptibility to epileptic seizures (Scharfman et al.

2000), blockade of NMDA receptors might counteract this develop-

ment in addition to its neuroprotective effects.

Acknowledgements

This work was supported by the Swedish Medical Research Council, SyskonenSvensson, Kock, Crafoord, and Elsa and Thorsten Segerfalk Foundations, theArbetsmarknadens FoÈrsaÈkringsaktiebolag, the Swedish Stroke Foundation, andthe Swedish Association of Neurologically Disabled.

Abbreviations

AMPA, (a-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid; BDNF,brain-derived neurotrophic factor; bFGF, basic ®broblast growth factor; BrdU,5-bromo-2¢-deoxyuridine-5¢monophosphate; DG, dentate gyrus; GCL, granulecell layer; MCAO, middle cerebral artery occlusion; MK-801, dizocilpinemaleate; NeuN, neuronal-speci®c antigen; NBQX, 2, 3-dihydroxy-6-nitro-7-sulfamoylbenzo(F)-quinoxaline; NGF, nerve growth factor; NMDA, N-methyl-D-aspartate; SD, standard deviation; SEM, standard error of themean; SGZ, subgranular zone.

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