Permanent or transient chronic ischemic

Journal of Cerebral Blood Flow & Metabolism (2010) 30, 273–285
& 2010 ISCBFM All rights reserved 0271-678X/10 $32.00
www.jcbfm.com

Permanent or transient chronic ischemic stroke in
the non-human primate: behavioral, neuroimaging,
histological, and immunohistochemical
investigations
Ebeline Bihel1, Palma Pro-Sistiaga2, Annelise Letourneur1, Jerome Toutain1,
Romaric Saulnier1, Ricardo Insausti2, Myriam Bernaudin1, Simon Roussel1
and Omar Touzani1
1

CERVOxy team, Hypoxia and cerebrovascular pathophysiology, CI-NAPS UMR-6232, CNRS, CEA,
Universite´ de Caen, Universite´ Paris Descartes, Caen, France; 2Human Neuroanatomy Laboratory, Department
of Health Sciences and CRIB, School of Medicine, University of Castilla-La Mancha, Albacete, Spain

Using multimodal magnetic resonance imaging (MRI), behavioral, and immunohistochemical
analyses, we examined pathological changes at the acute, sub-acute, and chronic stages, induced
by permanent or temporary ischemia in the common marmoset. Animals underwent either
permanent (pMCAO) or 3-h transient (tMCAO) occlusion of the middle cerebral artery (MCAO) by

the intraluminal thread approach. MRI scans were performed at 1 h, 8, and 45 days after MCAO.
Sensorimotor deficits were assessed weekly up to 45 days after MCAO. Immunohistological studies
were performed to examine neuronal loss, astrogliosis, and neurogenesis. Remote lesions were
analyzed using retrograde neuronal tracers. At day 8 (D8), the lesion defined on diffusion tensor
imaging (DTI)–MRI and T2-MRI was significantly larger in pMCAO as compared with that in the
tMCAO group. At D45, the former still displayed abnormal signals in T2-MRI. Post-mortem analyses
revealed widespread neuronal loss and associated astrogliosis to a greater extent in the pMCAO
group. Neurogenesis was increased in both groups in the vicinity of the lesion. Disconnections
between the caudate and the temporal cortex, and between the parietal cortex and the thalamus,
were observed. Sensorimotor impairments were more severe and long-lasting in pMCAO relative to
tMCAO. The profile of brain damage and functional deficits seen in the marmoset suggests that this
model could be suitable to test therapies against stroke.
Journal of Cerebral Blood Flow & Metabolism (2010) 30, 273–285; doi:10.1038/jcbfm.2009.209; published online
30 September 2009
Keywords: cerebral ischemia; chronic; functional recovery; model; MRI; non-human primate

Introduction
Many recent recommendations for preclinical investigations designed to develop stroke therapies call for
the use of higher order species such as non-human
primates in addition to rodent models (Fisher et al,

2009). These reviews also advocate the use of
multiple-outcome measures (functional deficits, neuroimaging along with post-mortem neuropathology)
both at the acute and the chronic stages of stroke to
assess the efficacy of an administered treatment. In
this context, we have recently described an original
Correspondence: Dr O Touzani, Centre Cyceron, UMR CI-NAPS
6232, Boulevard H Becquerel, BP 5229, Caen F-14074, France.
E-mail: touzani@cyceron.fr
Received 23 July 2009; revised 2 September 2009; accepted 8
September 2009; published online 30 September 2009

stroke model in the common marmoset (Freret et al,
2008), a New World monkey, in which middle cerebral artery (MCA) is occluded temporarily or permanently through use of an intravascular approach.
The common marmoset (Callithrix jacchus) may be
considered as the species of choice to study the
pathophysiology and the treatment of cerebral ischemia. Indeed, compared with rodents this primate is
closer to human in term of cerebrovascular system,
brain metabolism, gray-to-white matter ratio, and
rich behavioral repertoire. Weighing 250 to 400 g in
the adult age, the marmosets have a brain size approximately four times that of the rat, breed readily in

standard animal facilities, and are relatively easy to
handle, which is advantageous for behavioral testing
and postoperative care management (for review, see
Mansfield, 2003). The recent success in generating
transgenic marmosets has reinforced the value of

Chronic stroke in the marmoset
E Bihel et al
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using this species in modeling human diseases
(Sasaki et al, 2009).
In our recently published model of stroke in the
marmoset, brain damage as well as functional
impairments was lesser in marmosets subjected to
3-h temporary occlusion relative to those with
permanent MCA occlusion (MCAO). Although this
study proved the feasibility to induce a focal cerebral
ischemia in the non-human primate using the
intraluminal approach, the evaluation of damage

and behavioral deficits was restricted to the subacute
phase (i.e., 8 days after occlusion). Therefore, using
sequential magnetic resonance imaging (MRI), behavioral tests, histology, and immunohistochemistry,
we extended here the characterization of the model
and analyzed the evolution of cerebral damage and
behavioral impairments in acute, subacute, and
chronic stages of ischemia after permanent or temporary MCAO. We also examined neuronal loss,
astrogliosis, and neurogenesis associated to ischemia. Through use of neuronal retrograde tracers, we
extended the characterization of the model and
examined the degree of disconnections of remote
brain structures not affected by initial ischemia,
namely disconnection between the temporal cortex
and the striatum, and between the thalamus and the
parietal cortex. Both dorsal striatum and ventral
parietal cortex are known to have strong connections
with areas related to motor skills learning and of
sensory memory consolidation (Knowlton et al,
1996; Wise et al, 1996).

Materials and methods

All procedures were performed according to European
Directives (86/609/EEC) and approved by the Regional
Ethics Committee (agreement number 06-003).

Induction of Cerebral Ischemia
Six males and seven female laboratory-bred marmosets
(Callithrix jacchus, 285 to 370 g; aged 18 to 36 months) were
used. The animals were intubated and mechanically ventilated. Rectal temperature was kept stable with a heating pad.
The tail artery and vein were cannulated for arterial pressure
monitoring (Stoelting,Wooddale, IL, USA), blood sampling
for gases and pH analyses, and for atracurium (0.5 mg/kg; and
0.75 mg/kg/h; Tracrium, Faulding, Royal Leamington, UK)
and saline administration, respectively.
The details of the procedure of induction of ischemia
have been described previously (Freret et al, 2008). Briefly,
a nylon thread was inserted into the external carotid artery
and gently advanced up to the origin of the MCA. The
embolus was left in place in permanently occluded
marmosets (pMCAO, n = 5), removed after 3 h in transiently
occluded marmosets (tMCAO n = 6) or immediately in

Sham-operated animals (n = 2).
At the end of the experiment, animals received an
analgesic (tolfenamic acid, 4 mg/kg, intramuscularly; Tolfedine 4%; France) and the first dose of an antibiotic
Journal of Cerebral Blood Flow & Metabolism (2010) 30, 273–285

(cefamandole 15 mg/kg, intramuscularly; Kefandol, France;
two other doses were administered daily during the 2 next
days). After recovery from anesthesia, animals were
returned to their cages and provided access to water and
soft food, and observed for 1 h and daily.

Magnetic Resonance Imaging
The animals underwent three sequential MRI examinations
(7 T; Pharmascan, Bruker, France) at 1 h, 8, and 45 days
after MCAO. Each MRI session comprised echo-planar diffusion tensor imaging (DTI, 30 diffusion directions;
b = 1,000 and b = 0, echo time (TE)/repetition time
(TR) = 41.1/5,000 ms, Matrix M = 128  128, field of view
(FOV) = 50  50 mm, 10 slices, slice thickness = 1.5 mm),
time of flight angiography (TE/TR = 1.8/10 ms, M = 256 
192  96, FOV = 40  40  16.8 mm), T2*-weighted imaging

(TE/TR = 11/400 ms, M = 256  256, FOV = 50  50 mm, 20
slices, thickness = 1.5 mm), and fast T2-weighted imaging
(acceleration factor = 8, number of experiments = 4, TE/
TR = 60/5000 ms, M = 256  192, FOV = 50  50 mm, 20
slices, thickness = 1.5 mm). At day 8 (D8), a magnetization-prepared rapid acquisition gradient echo (MPRAGE)
sequence (TE/TR = 15/6000 ms, M = 256  256 FOV = 40 
40 mm, 16 slices, thickness = 0.7 mm) was performed
to calculate stereotaxic coordinates for neuronal tracer
injections.

Behavioral Assessment
The behavioral tests were performed in home cages
according to the procedure of Freret et al (2008), with
some modifications. The animals were trained on behavioral tasks during 1 week (W1) preceding MCAO,
evaluated on all tasks daily during the first week, and
weekly during 6 weeks by the same experimenter in a blind
manner.

Neurological Score
Absence (score = 2), moderate occurrence (score = 1), or

presence (score = 0) of the following abnormal movements
and postures were noted: forelimbs and hindlimb slipping
or dangling under the perch, at rest, or during movement;
hand crossing the chest; hindlimbs falling from the perch;
head tilting (before and after stimulation); and reaction to a
visual stimulus. A maximal score of 20 for each body side
was assigned.

Tactile Stimulation
The experimenter, without intruding into the visual field of
the animal, stimulates with a paintbrush each of its
forelimbs, hindlimbs, and ears. Scoring was performed as
follows: clear response = 2, moderate response = 1, or no response = 0 to assign a maximal score of 6 for each body side.

Chronic stroke in the marmoset
E Bihel et al

Hill-and-Valley Staircase
Marmosets must find and catch food rewards placed on
five steps of two staircases located behind a Plexiglas

screen attached to the front of the cage. In the hill version,
there are two laterally positioned vertical slots so that the
animal uses its right forelimb to retrieve the food reward
situated on the right stair, and vice versa. This test,
designed to study the motor impairment of each forelimb,
does not allow, however, the discrimination between
a motor deficit and an ipsilateral spatial deficit, which
was observed for some stroke models (Marshall and Ridley,
1996). As compared with the hill version, the valley
staircase dissociates the motor performances and the
visual field of the marmoset and, thus, prompts to
study the spatial exploration. Indeed, in the valley
version there is only one centrally positioned slot
and the animal uses its left forelimb to reach the
right stair, and vice versa. The maximal time to catch
all rewards was 300 secs for each version (Annett et al,
1994). A score was assigned for each step: 1 for the lowest
step to 5 for the upper step to yield a maximal score of
15 for each forelimb.

Six-Tube Choice
Six black plastic tubes were fixed to a Plexiglas strip,
which was placed horizontally at the front of the cage.
Marmosets could only reach the tubes if they stood on a
small central perch. The animal was required to search for
the reward located in one of the six tubes. The time to
reach the reward was recorded. Once the reward had been
retrieved or 30 secs had passed, the array of tubes was
removed and another tube was baited (Marshall et al,
2003b).

275

to an Olympus BX50 epifluorescence microscope (as
described by Mohedano-Moriano et al, 2005).

Histology and Immunohistochemistry
Forty-five days after MCAO, the marmosets were deeply
anaesthetized and transcardially perfused with a heparinized solution of saline followed by a solution of 4%
paraformaldehyde in phosphate buffer. The brains were

removed and cut in the coronal plane at 50 mm with a
freezing microtome (Microm, Heidelberg, Germany). One
section out of five was immediately mounted for thionin
staining. For immunohistochemistry, adjacent sections
were incubated with glial fibrillary acidic protein (GFAP)
(rabbit anti-GFAP 1:5,000; Dako, Trappes, France), NeuN
(mouse anti-NeuN 1:2,000; Millipore, Chandlers Ford,
Hampshire, UK), or tyrosine hydroxylase (TH) antibodies
(mouse anti-tyrosine hydroxylase 1:500; Millipore, Chandlers Ford, Hampshire, UK). Secondary biotinylated antirabbit 1:500 (Sigma-Aldrich, France) and biotinylated antimouse antibody 1:500 (Sigma-Aldrich, Saint-QuentinFallavier, France) were used.
To examine neurogenesis, the marmosets received a
daily injection of 5-bromo-2-deoxyuridine (BrdU, intraperitoneal (50 mg/kg); Sigma-Aldrich, France) from D1 to D18
after MCAO. For double BrdU + NeuN immunostaining,
adjacent sections were incubated with BrdU primary
antibody (rat anti-BrdU, 1:100; Abcam, Paris, France) and
NeuN primary antibody (mouse anti-NeuN 1:2,000; Millipore, Chandlers Ford, Hampshire, UK). Secondary antibodies were anti-rat Alexa 488 and anti-mouse Alexa 555
(1:200; Invitrogen, Cergy-Pontoise, France). For quantification of neurogenesis, the zones of interest were photographed and double labeled cells were counted and
expressed as a number of double labeled cells per square
millimeter.

Adhesive Removal
An adhesive label was placed around each hindlimb
(Annett et al, 1994). The order and latency to contact,
and the latency to remove the label, were measured
(600 secs maximum).

Neuronal Tracer Injections
At 30 days after the occlusion, one animal of each group
was placed in a stereotaxic frame and burr holes were
drilled at the intended injection sites. Pressure injections
of retrograde neuronal tracers Diamidine Yellow (DY; 2%;
Sigma-Aldrich, Madrid, Spain) and Fast Blue (FB; 3%,
Polysciences, Eppelheim, Germany) were performed in the
head of the caudate (antero-posterior = 11.5 mm; lateromedial = 3.4 mm; dorso-ventral = 5.1 mm from the interauricular stereotaxic plan) and the ventral parietal cortex
(antero-posterior = 11.5 mm; latero-medial = 10.3 mm; dorso-ventral = 1 mm), respectively (defined by MRI exam at
8 days with ImageJ software coordinate analysis). Analyses
of the retrograde tracers transport were realized by
counting positively labeled cells using a charting system
(MD3-Digitizer; AccuStage, Shoreview, MN, USA) adapted

Data Analysis
To identify areas with an abnormal signal, MR images were
thresholded at the mean±twice the standard deviation
(s.d.) of the contralateral values (ImageJ).
The data are presented as mean±s.e.m. Scores are
expressed as median±quartile. Mann–Whitney, Wilcoxon,
and Spearman tests were used where appropriate (Statview
software). A P < 0.05 was considered significant.

Results
Physiological Parameters

During the entire period of experiment, mean
arterial pressure (pMCAO: 68±3 mm Hg, tMCAO:
70±5 mm Hg, Sham: 75±2 mm Hg), heart rate
(pMCAO: 237±32 min 1; tMCAO: 230±30 min 1,
Sham: 239±79 min 1), and rectal temperature
(pMCAO: 37. 5±0.21C; tMCAO: 37.5±0.21C, Sham:
37.3±0.31C) remained stable within physiological
limits. Values for blood gases and pH were not
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276

different between the groups studied and remained
stable (for pMACO, tMACO, and Sham groups,
respectively, arterial CO2 tension was 42.5±
21.1 mm Hg, 42.3±14.2 mm Hg, and 42.4±12.3
mm Hg; arterial O2 tension was 176.3±21.4 mm Hg,
174.99±21.4 mm Hg, and 178.2±12.6 mm Hg; and
blood pH was 7.384±0.1, 7.427±0.1, and 7.431±0.1).
All the animals terminated the whole experimental protocol and no postoperative mortality was
observed in our experiments.

tMCAO: 19±1 at D1). However, all marmosets
exhibited long-lasting neurological impairment
(pMCAO: score = 2±1 at D1 and 8±0 at D41,
Wilcoxon test P = 0.027 and P = 0.025 and tMCAO:
score = 6±1 at D1 and 16±0 at D41, Wilcoxon test
P = 0.042 and 0.039). This deficit was more prominent and more durable for pMCAO as compared with
that for tMCAO (Figure 2A).

Tactile Stimulation
Magnetic Resonance Imaging

MR angiography showed disappearance of the MCA
in pMCAO at all the time points analyzed. Reperfusion was also confirmed by this approach in tMCAO
(Figure 1A).
At 60 mins after MCAO, a marked decrease in
apparent diffusion coefficient (ADC) was observed
mainly in the subcortical structures (Figure 1A).
The volume of tissue displaying these changes
was not different between pMCAO and tMCAO
(208.0±58.2 mm3 and 298.5±67.5 mm3 for pMCAO
and tMCAO, respectively; Mann–Whitney test
P = 0.23). At this time, no abnormal signal was
observed on T2-MRI (Figure 1A). At D8, the total
volume of lesion and the cortical lesion, measured
on either DTI or T2-MRI, were significantly higher
in the pMCAO as compared with that in the tMCAO
group (Mann–Whitney test P < 0.05; Figure 1B).
In addition, an abnormal signal was observed in
the substantia nigra at D8 (6.5±2.2 mm3 and
3.3±0.6 mm3 for pMCAO and tMCAO, respectively;
Figure 1C). The volume of this lesion was correlated
to the volume of abnormal signal in the striatum
(Spearman test, P = 0.0037; R = 0.918).
At D45, the area with an abnormal signal, on either
DTI or T2-MRI, decreased remarkably in the pMCAO
group (21.5±16.7 mm3) and completely resolved in
the tMCAO group. However, both groups of animals
showed atrophy of the ipsilateral hemisphere relative to the contralateral one (pMCAO: 4.8±1.7% and
tMCAO: 3.1±1.5%, Wilcoxon test P < 0.05). In all
animals, T2*-MRI did not reveal any abnormal signal
during the entire period of examination (Figure 1A),
suggesting absence of obvious hemorrhage. For
Sham-operated animals, no abnormal signal was
observed on all the imaging modalities at any of the
time points analyzed.
Behavioral Assessments

Before MCAO, all the animals showed no deficit on
behavioral tasks. Sham-operated animals also showed
no deficit during the entire period of investigation.
Neurological Score

After induction of ischemia, no animal showed
significant ipsilateral deficit (pMCAO: 18±1;
Journal of Cerebral Blood Flow & Metabolism (2010) 30, 273–285

At 1 day after ischemia, a decrease of contralateral
tactile perception was observed. Although this
deficit was long lasting for both groups (pMCAO
score = 0±1 at D1 and 1±1 at D41, and tMCAO
score = 1±1 at D1 and 4±4 at D41; Wilcoxon test
P < 0.05 as compared with D1), the pMCAO group
presented a stronger deficit as compared with the
tMCAO group at all the time points analyzed
(Mann–Whitney test P < 0.05 for comparison between pMCAO and tMCAO; Figure 2B). The animals
subjected to transient ischemia presented with
partial recovery from D8 (Wilcoxon test P = 0.026 as
compared with D1).

Hill-and-Valley Staircase

Before ischemia, all the marmosets completed the
two forms of the staircase test without significant
mistake and no difference in performances between
the right and left forelimbs was observed. Nonetheless, 1 week (W1) after ischemia, the animals
showed difficulties to catch the reward with the
contralateral forelimb (Figure 3B). This deficit was
bilateral at W1 for pMCAO group (Figure 3A). Ipsilateral deficit totally recovered at W2 and persisted
for the contralateral forelimb (score = 0±0 at W1,
P = 0.001 and score = 4.2±0.8 P = 0.042 at W7
(Wilcoxon test) as compared with D-1).
For the tMCAO group, the deficit was observed
until W5 (score = 7±1 at W1 P = 0.02, 11.9±1 at W4
P = 0.04, Wilcoxon test, compared with D1). Indeed,
partial recovery was observed for tMCAO since
W4 (P = 0.04 Wilcoxon test, compared with W1).
This deficit was more severe in the pMCAO group
during the time of observation (Mann–Whitney test
P < 0.05 for comparison between pMCAO and
tMCAO; Figure 3B).
As in the hill staircase test, the valley version
revealed a contralateral motor deficit, which was
more pronounced and durable for the pMCAO group
as compared with the tMCAO group (Figure 3C). In
addition, the valley staircase revealed a contralateral
perception deficit (Figure 3D). Indeed, we observed
significant decrease in the ipsilateral forelimb
score (i.e., contralateral space) (score = 10.5±2.6
and 13.1±0.2 at W2, and Wilcoxon test P = 0.04
and P = 0.04 as compared with D1 for pMCAO and
tMCAO, respectively). For the two groups of animal,

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E Bihel et al
277

Diffusion
tensor imaging

T2-weighted
imaging

T2*-weighted
imaging

Angiography
MCA

60 min

tMCAO

D8

D45

60 min

pMCAO

D8

D45

700

Lesion volume (mm3)

600

T2-weighted imaging

pMCAO
tMCAO

*

500

pMCAO

*

tMCAO

400
300
200
100
0
60 min
post-MCAO

total

cortex

subcortex

D45
post-MCAO

D8 post-MCAO

Figure 1 (A) Representative images of ADC maps obtained by DTI, T2-weighted imaging, MR angiography, and T2*-weighted
imaging at 60 mins, D8, and D45 after permanent MCAO (pMCAO) and 3-h temporary MCAO (tMCAO) the abnormal signals are
indicated by arrows. (B) Evolution of brain damage volume (mean±s.e.m.) measured on T2-MRI images. *P < 0.05 between
tMCAO and pMCAO groups. (C) Example of abnormal signal observed in the substantia nigra on T2-weighted imaging at 8 days after
MCAO for one animal in each group (arrows indicate the abnormal signal in the substantia nigra).
Journal of Cerebral Blood Flow & Metabolism (2010) 30, 273–285

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278

Score /20

A

secs and tMCAO time = 194.9±78.2 secs; Wilcoxon
test P < 0.05 as compared with W1).

$
20
18
16
14
12
10
8
6
4
2
0

*

D-1 D1

*

D2

*

*

*

*
#

*
#

*
#

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D3

D6

pMCAO
tMCAO

D8 D13 D20 D27 D41

D7

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B
6

*
#

5
Score /6

#

#

*

4
3

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2

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*

*

*

D1

D2

D3

D6

*
#

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1
0

D-1

D8 D13 D20 D27 D41

Figure 2 Evolution of contralateral neurological score (A) and
tactile stimulation score (B) in the pMCAO (n = 5 black bars)
and tMCAO (n = 6, gray bars) groups (median±quartile).
*P < 0.05 for difference between groups (Mann–Whitney test);
$
P < 0.05 in comparison with D1 indicating presence of deficit
(Wilcoxon test); and #P < 0.05 in comparison with D1 indicating recovery (Wilcoxon test). Dotted line represents the
performances of Sham-operated animals.

this deficit recovered at W3 after the induction
of ischemia.
Adhesive Removal

After MCAO, a contralateral tactile deficit was
measured in both groups (time = 600±0 and 268.6±
92.7 secs, Wilcoxon test P = 0.04 and P = 0.02, respectively, for pMCAO and tMCAO), with partial recovery from the fourth week (W4) (time = 404.4±220.9 s
at W6 and time = 67.3±22.7 s at W4, Wilcoxon test
P = 0.04 and P = 0.02 as compared with D1, respectively, for pMCAO and tMCAO). This deficit was
more severe for pMCAO (Mann–Whitney test,
P < 0.05 at W2 until W5).
A bilateral motor coordination deficit was also
evidenced. The time spent to remove adhesive labels
was increased as compared with pre-operative
measurements (W1: time = 600±0 and 466.7±
107.9 secs, respectively, for pMCAO and tMCAO for
the contralateral side, and time = 573.8±238.1 and
457.9±111.8 secs, respectively, for pMCAO and
tMCAO for the ipsilateral side).
In the ipsilateral side, we observed a partial
recovery from W5 for the two groups (pMCAO
time = 298.2±149.1 secs and tMCAO time = 194.9±
78.2 secs, Wilcoxon test P < 0.05 as compared with
D1) and in the contralateral side from W4 for tMCAO
and W5 for pMCAO (pMCAO time = 298.2±149.1
Journal of Cerebral Blood Flow & Metabolism (2010) 30, 273–285

Six Tubes Choice

Ischemia induced a deficit of spatial exploration as
the animals spent more time to find the rewards
located in the contralateral space (i.e., tubes 4 to 6).
As shown in Figure 5B, at 1 week after MCAO, the
marmosets subjected to temporary MCAO showed
contralateral alteration of visual perception (W1/W2:
time = 11.9±3.9 secs for tube 6; Wilcoxon test
P = 0.02 as compared with D1), which was transient
so as at W3; times to retrieve the rewards were not
different as compared with performances before
ischemia (Figure 4C and 4D, Wilcoxon test, P = 0.61
as compared with D-1 for tube 6). For animals
subjected to permanent MCAO, bilateral visual
perception deficit was observed during the first 2
weeks after the ischemia (Figure 4B). However, these
animals displayed only a contralateral hemineglect, which was long lasting (W5/W7: time = 11.92±
2.08 secs for tube 6; Wilcoxon test P = 0.04 as
compared with D-1). When the two groups of
animals were compared, the hemineglect, revealed
by this test, was more severe for the pMCAO group
during W4/W5 and W5/W7 (Mann–Whitney test
P < 0.05 for comparison between pMCAO and
tMCAO for tubes 5 and 6).

Correlation of the Functional Impairment with
the Extent of the Lesion

To determine whether the brain damage observed at
8 days after MCAO was predictive for behavioral
deficits, we performed correlation analyses between
the behavioral data obtained at the same period
and the lesion volume measured by DTI-MRI and
T2-MRI. The volume of subcortical damage measured with T2-MRI was correlated with the score of
the contralateral forelimb at the valley-and-hill
staircase tests 2 weeks after the occlusion
(P = 0.049, R = 0.6 and P = 0.02, R = 0.7, respectively, for the valley and hill staircase). Similarly,
there was a correlation with the deficit observed in
the tactile stimulation test at D8 (P = 0.04, R = 0.6)
and in the adhesive removal test at W2 (time to
contact the contralateral label, P = 0.017, R = 0.68).
The volume of cortical damage was correlated with
the degree of deficit as measured in the valley
staircase (ipsilateral score = contralateral hemispace:
P < 0.001, R = 0.95 and contralateral forelimb score
P = 0.02, R = 0.67) and the hill staircase tests
(contralateral forelimb P = 0.02, R = 0.66) at 2 weeks
after occlusion.
The volumes of the lesion determined with DTIMRI at D8 also correlated with the behavioral data,
although the statistical significance was weaker as
compared with that obtained with T2-MRI results.

Chronic stroke in the marmoset
E Bihel et al
279

Hill staircase

pMCAO
tMCAO

Contralateral forelimb

*

A
17
15

*

*

*

13

7

$

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5

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$

3
1
0

15

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9

$

$
D-1

W1

W2

*

17

$

$

11

B

#

Score /15

Score /15

13

*
#

#
$

Ipsilateral forelimb

11
9
7

$

5
3

W3

W4

W5

1
0

W7

D-1

W1

W2

W3

W4

W5

W7

Valley staircase

pMCAO
tMCAO

Contralateral forelimb
17

*

*

*

*

*

15

Score /15

$#

$

13

$

11

$

7

$

3

$

$

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W1

17

$

$ $

W1

W2

13
11
9

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7
5
3

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1
0

D-1

Ipsilateral forelimb

*

15

$ #

$#

$

9
5

D

Score /15

C

W2

W3

W4

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W7

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0

D-1

W3

W4

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W7

Figure 3 Evolution of performances in the hill (A and B) and valley (C and D) staircase tasks for the pMCAO (n = 5, black bars) and
tMCAO (n = 6, gray bars) groups (median±quartile). (A and C) Scores to retrieve rewards with the contralateral forelimb. (B and D)
Scores to retrieve rewards with the ipsilateral forelimb. *P < 0.05 for difference between groups (Mann–Whitney test); $P < 0.05 in
comparison with D1 indicating presence of deficit (Wilcoxon test); and #P < 0.05 in comparison with W1 indicating recovery
(Wilcoxon test). Dotted line represents the performances of Sham-operated animals.

Remote Injury

The sites of tracer’s injection were similar in each
group. The injection of DY into the caudate nucleus
revealed a reduction of labeled neurons in the
temporal cortex of occluded animals in comparison
with Sham-operated animal (Figure 5A and 5B), the
decrease being more pronounced in the pMCAO
animal. Similarly, the injection of FB into ventral
parietal cortex showed a decrease of labeled neurons
in the ipsilateral thalamus of animals subjected to

ischemia as compared with that in Sham-operated
animal, without major difference between pMCAO
and tMCAO (Figure 5C).
Histology and Immunochemistry

At 45 days after MCAO, thionin staining showed
both an increase in staining located in the caudate
nucleus and the putamen, and a lack of staining in
the cortex at the lateral fissure cortex (Figure 6A).
Journal of Cerebral Blood Flow & Metabolism (2010) 30, 273–285

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pMCAO
tMCAO
W1/W2

D-1

$

30

35

25

30

20
15
10
5
0

1

2

5

3
4
Tube number

Ipsilateral space

6

*

*

B

Mean cumulative time (s)

Cumulative time (s)

A 35

$

15

$

10
5
1

2

3
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Tube number

D

*

30

$

#
25
20
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#
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$
#

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$

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#

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0

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2

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W5/W7
35

$

6

Tube number

Mean cumulative time (s)

Mean cumulative time (s)

*

$

20

W3/W4

*

$

25

Contralateral space

35

*
$

$

0

C

*

$

30
25

*

20

#
$

15

$

#
10
5
0

*
#

#

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Tube number

#
#

5

6

Figure 4 Six-tube search task, illustrated in panel A, with results shown as latency (±s.e.m.) to find food reward hidden in 1 of 6
tubes positioned from marmoset’s far right (tube 1) to their far left (tube 6) by pMCAO (n = 4, black bars) and tMCAO (n = 4, gray
bars) marmoset before surgery; (B) mean 1 and 2 weeks after MCAO; (C) mean 3 and 4 weeks after MCAO; and (D) 5 and 7 weeks
after MCAO. *P < 0.05 for difference between groups (Mann–Whitney test); $P < 0.05 in comparison with D1 indicating presence of
deficit (Wilcoxon test); and #P < 0.05 in comparison with W1/W2 indicating recovery (Wilcoxon test). Dotted line represents the
performances of Sham-operated animals.

The increase in thionin coloration corresponded to
reactive gliosis as shown by GFAP immunostaining
(Figure 6C). Moreover, using a specific neuronal
marker, NeuN, widespread neuronal loss in caudate,
putamen, parietal, and temporal cortices (Figure 6B)
was observed. The use of tyrosine hydroxylase (TH)
Journal of Cerebral Blood Flow & Metabolism (2010) 30, 273–285

immunostaining showed a decrease of labeling in the
substantia nigra, confirming the changes seen
on MRI at D8 (Figure 6D). All of these changes
were more pronounced in permanently occluded
marmosets relative to those subjected to transient
ischemia.

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E Bihel et al
281

SHAM

B
Number of positive cells

900

pMCAO

848

800
700
600

C 1400

tMCAO
SHAM

1200

532

500
400
300
200

tMCAO

pMCAO

138

Number of positive cells

A

1285
1051

1000
800
602

600
400
200

100
0
Temporal lobe cortex

0
Thalamus

Figure 5 (A) Illustration of cell labeling in the temporal cortex after DY injection in the dorsal caudate nucleus (arrows) in three
marmosets (Sham, pMCAO, and tMCAO). Quantification of retrograde tracer transport from the caudate nucleus to the temporal lobe
cortex (B), and from the parietal cortex to the thalamus (C), after DY and FB injections in dorsal caudate nucleus and ventral cortex,
respectively. The data are shown as mean number of positive cells per slice labeled.

BrdU/NeuN labeling revealed in both groups the
existence of an ischemia-induced increase in neurogenesis in the ischemic boundary zone between the
subventricular zone and the striatal lesion, as well as
in the caudate, putamen, and the cortex, but not in
the dentate gyrus (Figure 6E). Although these BrdU/
NeuN labeling were more pronounced for the
marmosets subjected to ischemia (Mann–Whitney
test P = 0.06 as compared with Sham-operated), there
was no difference between temporarily and permanently occluded animals.

Discussion
The major finding in this study is that the intraluminal approach to induce cerebral ischemia in

common marmosets consistently results in cortical
and subcortical brain damage, evidenced by MRI,
histology, and immunohistochemistry, as well as in
long-lasting functional deficits, which are reduced
by reperfusion instituted as late as 3 h after the
occlusion. This makes this model suitable for testing
therapeutic interventions for stroke. The importance
of employing non-human primates in the study of
the pathophysiology and the treatment of brain
ischemia has been highlighted in many recent
reviews (Fisher et al, 2009, Gladstone et al, 2002,
STAIR, 1999). In this context, although marmosets
are not so close phylogenetically to human as
compared with old word monkeys (i.e., Cercopithecidae), they present several advantages over
both rodents and large primates (e.g., macaques
and baboon), making them potentially valuable in
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SHAM

pMCAO

tMCAO

Thionin

A

10 mm

NeuN

B

1mm

GFAP

C

TH

D

Number of BrdU+/NeuN+-cells/mm2

E

4

pMCAO

3.5

tMCAO
SHAM

3
2.5
2
1.5
1
0.5
0

contra

ipsi

contra

IBZ

F

ipsi

caudate putamen Lateral fissure cortex
superior inferior
Lesion area

DG

BrdU

NeuN

10µm

BrdU/NeuN

10µm

10µm

Figure 6 Representative photomicrographs illustrating thionin staining (A) and immunostaining in the head of caudate with NeuN
(B) and GFAP (C), and with tyrosine hydroxylase (TH) in the substantia nigra (D) in one animal per group. (E) Quantification of double
labeling (NeuN/BrdU-positive cells) is represented as mean (±s.e.m.) of double labeled cells per square millimeter per structure.
IBZ, ischemic boundary zone; DG, dentate gyrus (pMCAO (n = 4), tMCAO (n = 4), and Sham (n = 2)). (F) Illustration of BrdU-,
NeuN-, and BrdU/NeuN-positive cells in the IBZ (arrows indicate a BrdU/NeuN-positive cell).

Journal of Cerebral Blood Flow & Metabolism (2010) 30, 273–285

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modeling stroke and for testing stroke therapies
(Freret et al, 2008; Marshall and Ridley (1996);
Marshall et al, 2003a; Virley et al, 2004). We have
recently shown the feasibility to induce focal
cerebral ischemia by the intraluminal approach in
the common marmoset. This method has several
advantages over the previously described models in
this species: (1) the strict control of ischemia
durations; (2) the absence of craniotomy with
subsequent preservation of intracranial pressure
and brain temperature; (3) the simplicity of the
surgical procedure, which resembles the procedure
widely performed in rodents in many laboratories;
and (4) the comparative non-invasiveness as severe
disability and mortality was not observed in the
postoperative period. Overall, this approach allowed
us to perform behavioral and neuropathological
analyses in the subacute stage of infarction.
The use of multiple MRI modalities is important to
understand the evolution of the ischemic lesion. At
1 h after MCAO, DTI-MRI showed reduction of ADC
values especially in the subcortical structures. Tissue
volumes affected by these changes were similar for
both groups of marmosets, reflecting a similar degree
of the initial blood flow reduction in those animals.
At this time, and in accordance with the literature,
T2-MRI did not show any abnormal change since
vasogenic edema is not yet established. However,
8 days after MCAO, both T2-MRI and DTI-MRI
revealed the presence of abnormal signals that were
volumetrically reduced in the tMCAO group, indicating that restoration of blood flow as late as 3 h
after MCAO could reduce the brain lesion, especially
in the cortex. These findings imply that in this model
of stroke, the lesion evolves at least during 3 h after
the insult, an evolution closer to what is observed
for human relative to rodents (Touzani and Baron,
2007). Although the absolute ADC values are
comparable between marmosets (455±36 and
756±25  10 6 mm2/s for the lesion area and the
contralateral hemisphere, respectively), rodents
(486±62 and 716±58  10 6 mm2/s in lesioned and
contralateral striatum; Wang et al, 2003), and
humans (436±72 and 742±144  10 6 mm2/s; Eastwood et al, 2003), the profile of evolution of ADC
abnormalities observed in our study concords more
with that reported for humans for which the ADC
values were diminished during 10 days after the
ictus and reversed thereafter (Eastwood et al, 2003;
Munoz Maniega et al, 2004). In contrast, the acute
reduction of ADC values for rodents normalizes in
general at 4 days after the occlusion (NeumannHaefelin et al, 2000). The diminished ADC observed
at 8 days after MCAO proved that the time course of
cerebral damage in the marmoset was closer to
human pathology than rodent pathology. This relative slow evolution of the lesion, as compared with
that for rodents, could be explained by the existing
differences in neuronal and glial densities and
white-to-gray matter ratio, and, thus, basal brain
metabolism between rats and primates (Kennedy

et al, 1978). In accordance with our previous study
(Freret et al, 2008), T2*-MRI showed absence of
hemorrhage confirming that introduction of the
filament did not damage the vessel, and absence of
hemorrhagic transformation in the sub-acute and
chronic stage. The absence of micro-hemorrhage in
this model was also confirmed by histological
analyses (Perls coloration) at 8 days after stroke
(Freret et al, 2008).
In the chronic stage, both DTI and T2-MRI showed
apparent reduction of the lesion, which was more
pronounced in tMCAO. This profile of evolution that
depends on the duration and severity of the
ischemia, has been reported in many studies both
of rodents and primates (Esneault et al, 2008;
Wegener et al, 2006, Hirouchi et al, 2007), and
reflects the occurrence of inflammation, glial reaction, and reorganization of the affected hemisphere
at the chronic stage. This reorganization gives place
to shrinkage of the ipsilateral hemisphere as observed
in our study. Cavitation formation was observed only
in some pMCAO marmosets, which were, however,
smaller to that reported by Marshall et al (2003b)
in a model of permanent extraluminal occlusion of
the MCA in the marmoset, a model in which almost
the entire hemisphere is damaged.
In this study, based on the use of a battery of
sensorimotor tests, we have assessed the evolution of
functional deficits during 45 days after MCAO. We
have demonstrated the presence of hemiparesis,
hemianesthesia, and hemineglect in both groups. In
general, the contralateral deficits were more severe
and long lasting in the marmosets subjected to
pMCAO relative to those subjected to tMCAO. The
time course of the deficits seen in pMCAO was
globally in agreement with that reported by Marshall
and Ridley (1996). But the long-term evolution of
deficits in tMCAO was not described previously.
Here we have shown a long-lasting neurological
impairment in this group, although the animals
displayed better spontaneous recovery. Nonetheless,
we observed a bilateralism of deficits in some tests
such as the hill staircase during the first week. This
can be attributed to the presence of nystagmus and
spontaneous rotations at this time and prevented
marmosets to focus on the task. These spontaneous
rotations also explain the bilateral deficits shown in
pMCAO in the six tubes task. In the adhesive
removal task, we also observed an increase of time
to remove the adhesive from the ipsilateral hind
limb, which can be explained by the presence of
contralateral hemiparesis, which makes it difficult
for the animal to use its affected limbs to remove the
adhesive label in the non-affected ones. In general,
the functional deficits seen in this model both in the
acute and the chronic stages of ischemia were in
accordance with the situation observed in man
(Feigin et al, 2003; Varona et al, 2004).
Post-mortem analyses confirmed the reorganization of the ipsilateral hemisphere. Indeed, thionin
staining revealed the presence of cavitation in some
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284

animals in the pMCAO group and cellular loss
especially in the parieto-temporal cortex around the
lateral fissure. In both groups, and in contrast to what
we observed at 8 days after MCAO (Freret et al,
2008), thionin staining also elicited an increase of
signal in the striatum and the frontoparietal cortex,
reflecting the high cellular density that corresponds
to reactive gliosis. The latter was indeed confirmed
by GFAP staining that closely matched the area with
increased thionin labeling. The use of a specific
neuronal marker, NeuN, revealed neuronal loss in
the striatum and the cortex, which was more
prominent in pMCAO relative to tMCAO.
Additionally, TH labeling revealed neuronal loss
located in the substantia nigra, which confirmed the
MRI observations at 8 days. This substantia nigra lesion
has been described previously in rat and macaque
after chronic stroke (Abe et al, 2003; Hirouchi et al,
2007), and described retrograde degeneration of
nigro-striatal neurons. In our study, we found also
an increase in GFAP labeling, confirming a glial
reaction associated to the substantia nigra lesion, but
the significance of this remote lesion in the evolution
of ischemia-induced deficit is not established.
The analysis of double NeuN + BrdU immunostaining allowed the quantification of the neurogenesis in
our model. The effect of age on neurogenesis in the
marmoset’s brain was previously described by
Leuner et al (2007), but this phenomenon has never
been described under pathological conditions. It is
widely accepted that ischemia increased neurogenesis, and that new neurons migrate from neurogenic
regions (subventricular zone) to the infarct area (for
review, see Zhang et al, 2008). In this study, we
observed, for the first time, increase of neurogenesis
in marmosets after stroke as reported for rodents.
This increase was observed in caudate and putamen
nuclei, and in the lateral fissure cortex and ischemic
boundary zone, but not in the hippocampus. This
observation is of interest since this model of stroke
could also be employed in studies aiming to test
therapeutic strategies designed to increase neurogenesis. Overall, these data emphasize the importance of
using different markers of cellular loss and cellular
proliferation to evaluate the amplitude and the type
of tissue changes during the maturation of the
ischemic lesion.
Although remote injuries should not be claimed
based solely on the neuronal tracer results, we
showed that neuronal disconnections between remote brain structures are not affected by the initial
ischemia and the lesion areas. The use of FBretrograde neuronal tracer allowed us to show an
alteration of neuronal thalamic projections to the
ventral parietal cortex, a zone perfused by the MCA
and then affected by the ischemia as suggested for
humans (Nagasawa et al, 1994). These data are in
agreement with studies that described delayed
atrophy of the thalamus after MCAO in rodents and
humans, although in these studies no tracer injections where realized to confirm this disconnection
Journal of Cerebral Blood Flow & Metabolism (2010) 30, 273–285

(Freret et al, 2006; Nakane et al, 2002; Stebbins et al,
2008). Similarly, DY-retrograde neuronal tracer
revealed temporal cortex projections to caudate
nucleus as previously described in another healthy
non-human primate study (Selemon and GoldmanRakic, 1988). These projections were markedly
altered in animals subjected to MCAO, suggesting
retrograde degeneration after the lesion of the caudate (Nagasawa et al, 1994). Of note, this ischemiainduced alteration of these projections did not result
in any changes in MRI signal (both DTI and T2-MRI).
This suggests that the use of the neuronal tracer
approach would be more sensitive in depicting disconnections than MRI alone. Although these results
are preliminaries analyses, they evidence the presence of disconnections between structures in this
marmoset stroke model. The significance of these
disconnections for functional deficits is not completely known. Secondary thalamic and temporal cortex
atrophies could underlie, at least in part, sensorimotor and memory disturbances seen in patients.
In conclusion, through use of multiple analyses
with MRI, behavioral tests, histology, and immunochemistry, we show that focal stroke in the marmoset, induced by intraluminal occlusion of the MCA,
results in widespread brain damage and long-lasting
functional deficits that are reduced by reperfusion.
This makes this model suitable to test new therapies
against stroke.

Acknowledgements
This study was supported by the French Centre
National de la Recherche Scientifique (CNRS) and
the french conseil re´gional de Basse-Normandie. The
Ministry of Foreign Affairs (Spain) for the Picasso
project is also acknowledged.

Conflict of interest
The authors declare no conflict of interest.

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