Journal of the Neurological Sciences 347 (2014) 275280

Contents lists available at ScienceDirect

Journal of the Neurological Sciences

journal homepage: www.elsevier.com/locate/jns

Enriched environment induces angiogenesis and improves neural

function outcomes in rat stroke model
Kewei Yu a,b, Yi Wu a,b,c,d,e,, Qi Zhang a,b, Hongyu Xie a,b, Gang Liu a,c, Zhenzhen Guo a,b, Fang Li a,e, Jie Jia a,b,d,
Shenyi Kuang f, Ruiping Hu a,d,
a

Department of Rehabilitation, Huashan Hospital, Fudan University, Shanghai 200040, China

State Key Laboratory of Medical Neurobiology, Fudan University, Shanghai 200032, China
The Yonghe Branch of Huashan Hospital, Fudan University, Shanghai 200436, China
d
Department of Sports Medicine and Rehabilitation, Shanghai Medical College, Fudan University, Shanghai 200032, China
e
Department of Rehabilitation Medicine, Jing'an District Centre Hospital of Shanghai, 200040, China
f
Department of Clinical Medicine, Shanghai Medical College, Fudan University, Shanghai 200032, China
b
c

a r t i c l e

i n f o

Article history:
Received 12 June 2014
Received in revised form 18 September 2014
Accepted 13 October 2014
Available online 20 October 2014
Keywords:
Enriched environment
Ischemia
Neural function
CD31
Angiogenesis
Neuroprotection

a b s t r a c t
Increasing evidence shows that exposure to an enriched environment (EE) after cerebral ischemia/reperfusion
injury has neuroprotective benets in animal models, including enhancing functional recovery after ischemic
stroke. However, the mechanism underlying this effect remains unclear. To clarify this critical issue, the current
study investigated the effects of EE on the improvement of damaged neural function and the induction of
angiogenesis. Adult rats were subjected to ischemia induced by middle cerebral artery occlusion followed by
reperfusion. Neurological status scores were used to evaluate neural function on postoperative days 2, 7, and
14. A beam-walking task was used to test the recovery of motor behavior on postoperative days 2, 5, 10, and
15. We also used a Morris water maze task to examine whether EE protected learning and memory performance.
The specic marker of angiogenesis of CD31 was examined by western blot. Angiogenesis around the periinfarction region was assayed by laser scanning confocal microscopy (LSCM) after 14 days of EE exposure starting
24 h after ischemia. Neurological status scores of animals in the EE group were signicantly higher than those in
the standard housing condition (SC) control group from the seventh day after ischemic. EE accelerated the recovery
of motor coordination and integration and also improved learning and memory performance after cerebral
ischemia. Furthermore, EE increased CD31 levels and promoted angiogenesis of cortex in the peri-infarction
region compared to the SC group. Neural function outcomes are positively correlated with post-ischemia
angiogenesis. These ndings suggest that EE plays an important role in the recovery of damaged neural
function via regulation of angiogenesis after ischemia.
2014 Elsevier B.V. All rights reserved.

1. Introduction
Functional impairment caused by stroke is a highly serious health
problem throughout the world [1] and causes enormous burdens.
Therefore, it is extremely important to improve the functional recovery
of stroke patients.
Enriched environments have been used as a model of rehabilitation
in rodents. Enrichment entails housing multiple animals together in a
large cage equipped with different toys and with enhanced novelty
and complexity relative to standard conditions [1,2]. The benecial
effect of EE on stroke rehabilitation has been previously demonstrated
in animal models [3,4]. Recent increasing evidence has demonstrated
Corresponding authors at: Department of Rehabilitation Medicine, Huashan Hospital,
Fudan University, WuLuMuQi Middle Road 12, Shanghai 200040, China. Fax: + 86
2152887820.
E-mail addresses: doctoryu8000@163.com (Y. Wu), rphu79@163.com (R. Hu).

http://dx.doi.org/10.1016/j.jns.2014.10.022
0022-510X/ 2014 Elsevier B.V. All rights reserved.

that housing rats in an enriched environment after focal cerebral

ischemia substantially improves functional outcomes [5,1,6]. Yet while
the positive effects of EE on functional outcomes after stroke are widely
recognized, their underlying mechanisms are poorly understood.
Angiogenesis is one of the major styles of new vessel formation.
Cerebral angiogenesis after ischemia could be one promising strategy
for functional improvement after stroke [7,8]. Studies have shown that
stroke patients have reduced morbidity and longer survival time
because of a higher density of blood vessels [9,10]. Focal angiogenesis
may provide sufcient oxygen and nutrition for cerebral reconstruction
and may participate in remodeling the damaged area during the
subacute phase [11] so as to improve functional outcomes. Therefore,
angiogenesis might play an important role in the recovery of the neural
function of stroke patients.
The present study tested the hypothesis that EE can alleviate ischemic
damage and improve functional outcomes by increasing the emendation
of microvascular structure and by inducing angiogenesis. Specically, we

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investigated the effect of EE on behavioral status, angiogenesis, and

expression of CD31 after cerebral ischemic injury.

(30 cm wide 40 cm long 20 cm high) with no objects. The shamoperated animals were also housed in standard cages.

2. Material and methods

2.4. Neural function test

2.1. Animals

2.4.1. Neurological status evaluation

Neurological status was assessed on postoperative days 2, 7, and 14
in a blinded fashion using a seven-point scale [14] as follows: (0) no
decit; (1) failure to extend right forepaw fully; (2) decreased grip of
the right forelimb when held by tail; (3) spontaneous movement in all
directions, but torso turning to right when held by tail; (4) circling or
walking to the right; (5) walking only in response to tactile stimulation;
(6) no spontaneous activity; and (7) dead.

Healthy male SpragueDawley rats (Sino-British Sippr/BK Lab

Animal Ltd., Shanghai, China) weighing 250280 g were housed at
20 C on a 12 h light/dark cycle (lights on from 8:00 to 20:00) with
free access to water and standard rat chow. Animals were randomly
assigned to three experimental groups: ischemia with enriched environment (EE), ischemia with standard condition (SC), or sham surgery
with standard condition. Experiments were performed according to the
Guidelines of the National Institutes of Health Guide for the Care and
Use of Laboratory Animals. The protocol was approved by the Animal
Experimental Committee of Fudan University in Shanghai, China.
2.2. Middle cerebral artery occlusion (MCAO)
Ischemia was induced by left MCAO, as previously described [12].
Briey, rats were anesthetized with 10% chloral hydrate (350 mg/kg,
i.p.). Body temperature was maintained at 37 C throughout the procedure using a circulating heating pad. A 4-0 surgical nylon monolament
with a silicone tip was advanced from the external carotid artery into
the lumen of the internal carotid artery. After 90 min of cerebral
ischemia, the lament was withdrawn to allow reperfusion. In the
sham-operated control group, rats underwent the same surgical
procedure except for MCAO. Once animals recovered from anesthesia,
they were scored based on a four-point scale [13] as follows: (0) no
neurological symptoms; (1) unable to completely extend the front
contralateral paw; (2) rotating while crawling and falling to the contralateral side; (3) unable to walk without assistance; and (4) unconsciousness [did not recover]. Rats with scores of 13 points were
considered successful models and were included in the study. After
scoring, all rats were returned to SC home cages. After one day of
MCAO, rats in the EE group were returned to their EE home cages.
2.3. Housing conditions
Starting the day after surgery, we housed rats in the EE group in
enriched environment home cages (patent number of the People's Republic of China: ZL 2010 2 0560281.3). As shown in Fig. 1, EE housing
was larger (80 cm wide 120 cm long 100 cm high) than standard
housing, and contained climbing platforms, plastic tubes and tunnels,
chains, and small boxes. In addition, EE provided enhanced social stimulation because a total of 8 to 12 rats were group-housed, and animals
were changed every three days to maintain the novelty in the environment. SC rats were group-housed in sets of three in standard cages

2.4.2. Beam-walking test

We used a beam-walking task to assess decits in coordination and
integration of movement in the rats after ischemia, especially in
hindlimbs [15]. The beam-walking apparatus consisted of a square
beam (2.5 cm wide, 140 cm long, at a height of 42 cm) connected to a
black box (40 cm 20 cm 25 cm). The rats were trained for three
days to traverse the beam before ischemia induction and were tested
for beam-walk task performance the day before surgery to establish a
baseline measure. The animals were re-tested on postoperative days 1,
3, 5, 7, and 14. Their beam-walking performance was video recorded;
three trials were recorded for analysis. Performance was rated as
follows: rat was not able to stay on the beam (0 points); rat did not
move but was able to stay on the beam (1 point); rat tried to traverse
the beam but fell (2 points); rat traversed the beam with more than
50% footslips of the affected hindlimb (3 points); rat traversed the
beam with more than one footslip but less than 50% (4 points); rat
had only one slip of the hindlimb (5 points); and the rat traversed the
beam without any slips of the hindlimb (6 points) [16].
2.4.3. Morris water maze task
We used the Morris water maze task to test the spatial learning and
memory of the rats as described previously [17] with some modications.
Briey, the water maze was a black circular pool lled with water
(2123 C water temperature) and situated in a room with salient
visual cues. Spatial learning acquisition occurred during postoperative days 1619 for four consecutive days. Every rat received
8 consecutive trials (with randomly assigned starting positions)
per day to locate the platform that was submerged 2 cm beneath
the water surface. They were allowed 60 s to locate the platform,
which was kept in a constant location. If they failed to locate the platform within 60 s, rats were manually guided to the platform and left
there for 10 s. The mean escape latency per day was recorded for each
animal and used in statistical analysis. One day after the nal acquisition
training session (day 20 post MCAO), all rats performed a probe test
with the escape platform removed. Animals were placed into the pool

Fig. 1. EE and SC housing conditions.

K. Yu et al. / Journal of the Neurological Sciences 347 (2014) 275280

in the location most distal to the target quadrant (with the platform removed). The percent of time spent in the target quadrant was recorded
and interpreted as spatial memory [18].
2.5. Vascular labeling and analysis of vascular density
In order to examine functional vessels in the ischemic brain, we used
lectin, which selectively binds to endothelial glycocalyx only within
perfused vessels. Fluorescein isothiocyanate (FITC)-dextran (2 106
molecular weight, Sigma; 1 ml of 50 mg/ml) was administered intravenously to ischemic rats subjected to 15 days of tMCAO. This dye was
allowed to circulate for 1 min, after which the anesthetized animals
were killed by decapitation. Whole brains were quickly removed and
placed in 4% paraformaldehyde at 4 C for 48 h. Coronal sections
(50 m thick) were cut on a vibratome for each rat; ten coronal sections
of 1-mm intervals were obtained. The brain sections were analyzed
with a laser-scanning confocal imaging system. Eight brain regions in
the ischemic boundary zone were selected within a reference coronal
section (interaural 8.8 mm, bregma 0.8 mm) [19]. Vascular surface
area (mm2) was calculated by Image-Pro Plus software, and the number
of vascular branch points was counted in the microscope by a blinded
investigator.

277

3. Results
3.1. Neural function outcomes
3.1.1. Effect of EE on neurological status
Sham rats had no neurological symptoms as indicated by neurological status scores of zero. As shown in Fig. 2A, there was no difference
of neurological status between the EE group and the SC group on
postoperative day 2. However, EE rats showed signicant improvement
relative to SC rats (p b 0.05) on postoperative days 7 and 14.
3.1.2. Effect of EE on beam-walk task
A beam-walk task assesses the decits in coordination and integration
of motor movement in rats after ischemia. As shown in Fig. 2B, ischemic
rats with EE had better beam-walking performance as compared to SC
rats (p b 0.05).

Ipsilateral cortical tissue surrounding the ischemic zone was harvested 15 days after MCAO. Cortical protein extracts and western
blotting analysis were performed as previously described [20]. Protein
(40 g) was separated by 15% SDS-polyacrylamide gel electrophoresis
(SDS-PAGE) and transferred to PVDF membranes. The primary antibody
was anti-CD31 (Cell Signaling Technology). Anti-glyceraldehyde
3-phosphate dehydrogenase (GAPDH: Cell Signaling Technology)
was used as a loading control. Membranes were then washed and incubated with secondary antibodies for 1 h at room temperature. Quantication of band intensity (optical density) was carried out on scanned
western blot images using Image J software from blots of independent
experiments.

3.1.3. Effect of EE on spatial learning and memory

In addition to motor decits, cerebral ischemia is also known to impair spatial learning and memory depending on the location and severity of the insult [21]. We examined whether EE protected learning and
memory performance following ischemic insult using the Morris
water maze. The initial assessment of spatial learning, as quantied
using the metric of escape latency to nd the submerged platform, revealed that the three groups had no signicant difference on day 1
(day 16 post tMCAO), but rats in the SC group took signicantly longer
to nd the platform on days 2, 3, and 4 of training (days 17, 18, and 19
post tMCAO, respectively), when compared to both the EE and sham
groups (Fig. 3A). There was no statistical difference between the EE
and sham groups in spatial learning (Fig. 3A).
To evaluate spatial memory, we performed a probe trial on the day
after the last escape latency trial (i.e., day 20 post tMCAO). During this
trial, the platform was removed and the time rats spent in the quadrant
of the former platform was measured (correct quadrant). The results
indicated that rats in the SC group spent signicantly less time in the
correct quadrant compared to both the EE and sham groups (Fig. 3B).
There was no signicantly difference between the EE and sham group
(Fig. 3B).

2.7. Statistical analysis

3.2. EE enhances post-stroke revascularization in the ischemic boundary

zone

All data are expressed as mean S.E.M. Statistical analyses were

performed using SPSS 13.0 statistical software. We used one-way
ANOVAs followed by LSD multiple comparison post hoc tests to between groups. p values b 0.05 were considered to represent statistically
signicant differences.

The neuroprotective effect provided by EE was suggestive of true

neurological rehabilitation, which is likely to be associated with
angiogenesis. To test the hypothesis that post-stroke angiogenesis
is enhanced by EE, we next examined whether EE affected revascularization in the ischemic boundary zone. As shown in Fig. 4, the

2.6. Western blotting

Fig. 2. Neural function outcomes. (A) Neurological status. EE rats showed signicant improvements relative to SC rats on postoperative days 7 and 14. (B) Beam-walking test scores. Relative to SC controls, rats exposed to EE had signicantly higher neurological scores, indicating motor function improvement. Data are expressed as mean S.E.M. *p b 0.05 versus SC group,
#
p b 0.05 versus sham group.

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Fig. 3. Effect of EE on spatial learning and memory. (A) Evaluation of spatial learning using the Morris water maze latency test following tMCAO demonstrated that the three groups had no
signicant difference on day 16 post tMCAO, but rats in the SC group require more time to nd the platform on days 17, 18, and 19 post tMCAO, respectively, when compared to both the EE
and sham groups. There was no signicant difference between the EE and sham groups in spatial learning. (B) Determination of spatial memory using the probe test on day 20 post tMCAO.
Rats in the SC group spent signicantly less time in the correct quadrant compared to both the EE and sham groups. There was no signicant difference between the EE and sham groups.
Data are expressed as mean S.E.M. *p b 0.05 versus SC group, #p b 0.05 versus sham group.

vascular surface area and the number of vessel branch points were
higher in the EE group as compared to the SC group in the same
volume (p b 0.05).
3.3. Western blot analysis of CD31 expression
CD31 is the marker of angiogenesis. Therefore, we further assessed
CD31 levels in the ischemic boundary zone by western blotting. As
shown in Fig. 5, western blot analysis revealed increased CD31 protein
expression 15 days post MCAO in the EE group as compared to SC
group (p b 0.05), indicating that EE indeed promoted angiogenesis in
the ischemic boundary zone.
3.4. Correlation of neural function outcomes with angiogenesis
Finally, to assess a possible correlation between neurological function
outcomes and angiogenesis, we compared the neurological status scores
and cognitive function scores to the quantied rate of angiogenesis by

using Spearman's correlation coefcients (r). Correlations between

the neurological function and angiogenesis are shown in Fig. 6. The neurological status scores were negatively correlated with angiogenesis
(Fig. 6A, B) and cognitive functions were positively correlated with
angiogenesis (Fig. 6C, D). These results imply that neural function outcomes are correlated with angiogenesis.
4. Discussion
The current study is the rst to investigate the effects of EE on angiogenesis and how angiogenesis is related to improved function outcomes
in an ischemic rat model. EE increased CD31 levels and promoted angiogenesis of rat cortex in an ischemic rat model. EE also accelerated the recovery of coordination and integration of motor skills and improved
learning and memory performance. Furthermore, we found that neural
function outcomes are signicantly correlated with angiogenesis. Taken
together, these results suggest that EE might promote angiogenesis
after ischemic injury, thus improving neural function outcomes.

Fig. 4. EE enhanced post-stroke revascularization in the ischemic boundary zone. The top panels show representative images of lectin uorescent signal in the ischemic boundary zone of
brains at 15 days after tMCAO. Bottom panels of the gure demonstrate how the vascular surface area (A) and the number of vessel branch points (B) were higher in the EE group as compared to the SC group in the same volume. Data are expressed as mean S.E.M. *p b 0.05 versus SC group, #p b 0.05 versus sham group.

K. Yu et al. / Journal of the Neurological Sciences 347 (2014) 275280

279

Fig. 5. Western blot analysis of CD31 in the ischemic boundary zone at 15 days after tMCAO. Relative to the SC group, rats in the EE group had signicantly higher levels of CD31. Optical
density values normalized to their respective GAPDH loading control were mean SD and graphed (relative expression). *p b 0.05 versus SC group, #p b 0.05 versus sham group.

EE is well established to protect against ischemia-induced brain injury [22,2], especially in terms of improved function outcomes [23,24]. Previous studies have suggested that EE is able to induce neuroplastic
changes following stroke, including neurogenesis [25,26], synaptogenesis [27], increased dendritic branching and spine density [28,29] and
neurotrophins [30,31]. All of these above changes may contribute to
the recovery of function outcomes after ischemic injury. Simultaneously,
angiogenesis is essential for ischemic brain repair and function outcome
recovery because it stimulates blood ow and metabolism in the ischemic boundary [32]. This is because the most important pathological
event that contributes to the impairment of brain function in the ischemic brain is a shortage of glucose and oxygen supply. Neurons are highly
sensitive to hypoxiaischemia. However, focal angiogenesis may provide
sufcient oxygen and nutrition for cerebral reconstruction by neuronal
progenitor cell differentiation and migration during the post-ischemia

period [33]. Therefore, an efcient vascular supply of blood is capable

of preventing the progression of ischemic pathogenesis, including cerebral infarction and tissue degeneration, and may correlate with functional recovery after cerebral ischemia [34].
During the early acute phase of neurovascular injury, bloodbrain
barrier perturbations should predominate with key roles for various
matrix proteases. During the delayed phase, brain angiogenesis may
provide the critical neurovascular substrates for neuronal remodeling
[35]. In our research, we focus on the long-term neurological function
recovery, not the early acute phase of neurovascular injury. Together
with our results, EE improves neural function outcomes after cerebral
ischemia and promotes angiogenesis of the rat cortex in the ischemic
boundary zone. Neural function outcomes are positively correlated
with post-ischemia angiogenesis. Besides, one report has suggested
that increased angiogenesis is not associated with increased cerebral

Fig. 6. Correlation of neural function outcomes with angiogenesis. r represents Spearman's correlation coefcient. p b 0.05 was considered a signicant correlation.

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K. Yu et al. / Journal of the Neurological Sciences 347 (2014) 275280

edema formation as well as no further destruction of the blood brain

barrier [36]. Furthermore, clinical research has suggested that stroke patients with a higher density of blood vessels appear to have reduced
morbidity and survive longer [37]. Therefore, EE plays an important
and benecial role in the recovery of damaged neural function via regulation of angiogenesis after ischemia.
EE is a powerful tool to counteract cognitive and somatosensory deficits and is a broad noninvasive strategy in treating neurological disorders following brain injury. At present, no studies have yet examined
the effects of EE on angiogenesis after brain ischemia. CD31 (also called
PECAM-1) is expressed in all cells within the vascular compartment and
plays an important role in angiogenesis [38]. CD31 is considered a
marker of angiogenesis [39,40]. In our studies, we found that CD31 expression was dramatically increased and that there was more microvasculature in the peri-infarction region of the EE group as compared with
that in the SC group, suggesting that EE has can improve angiogenesis
after ischemia stroke. In addition, the neuron function outcome of the
EE group was better than the SC group after MCAO. Furthermore, we
found neural function outcomes are correlated with the level of angiogenesis. Taking all of these observations into account, we speculate
that behavioral improvement after brain ischemia is due to angiogenesis and that angiogenesis may be one of the recovery mechanisms that
EE induces following cerebral ischemia.
In conclusion, we have demonstrated that EE improves neural function outcomes after cerebral ischemia and promotes angiogenesis of the
rat cortex in the ischemic boundary zone. Although these results are encouraging in the study of stroke therapy, they still need to be conrmed
on a larger scale and our future research program will elucidate the
mechanisms of EE-induced angiogenesis.
Conicts of interest
There are no conicts of interest.
Acknowledgments
The present study is supported by the National Natural Science
Foundation of China (81171856, 81171855, 81401866), National Program on Key Basic Research Project of China (973 Program, no.
2010CB945500) and Major project of Shanghai Science and Technology
Commission (no. 13411951000).
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