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,b,

ity, Shanghai 200032, China

a r t i c l e i n f o

Article history:

function via regulation of angiogenesis after ischemia.

ne promising strategydies have shown thatlonger survival time0]. Focal angiogenesis

Journal of the Neurological Sciences 347 (2014) 275280

Contents lists available at ScienceDirect

Journal of the Neur

j ourna l homepage: www.eand complexity relative to standard conditions [1,2]. The benecialeffect of EE on stroke rehabilitation has been previously demonstratedin animal models [3,4]. Recent increasing evidence has demonstrated

may provide sufcient oxygen and nutrition for cerebral reconstructionand may participate in remodeling the damaged area during thesubacute phase [11] so as to improve functional outcomes. Therefore,of stroke patients.Enriched environments have been used as a model of rehabilitation

in rodents. Enrichment entails housing multiple animals together in alarge cage equipped with different toys and with enhanced novelty

Cerebral angiogenesis after ischemia could be ofor functional improvement after stroke [7,8]. Stustroke patients have reduced morbidity andbecause of a higher density of blood vessels [9,1 2014 Elsevier B.V. All rights reserved.

1. Introduction

Functional impairment caused by stroke is a highly serious healthproblem throughout the world [1] and causes enormous burdens.Therefore, it is extremely important to improve the functional recovery

that housing rats in an enriched environment after focal cerebralischemia substantially improves functional outcomes [5,1,6]. Yet whilethe positive effects of EE on functional outcomes after stroke are widelyrecognized, their underlying mechanisms are poorly understood.

Angiogenesis is one of the major styles of new vessel formation. Corresponding authors at: Department of RehabilitatiFudan University, WuLuMuQi Middle Road 12, Shang2152887820.

E-mail addresses: doctoryu8000@163.com (Y. Wu), rp

http://dx.doi.org/10.1016/j.jns.2014.10.0220022-510X/ 2014 Elsevier B.V. All rights reserved.ischemia. Furthermore, EE increased CD31 levels and promoted angiogenesis of cortex in the peri-infarctionregion compared to the SC group. Neural function outcomes are positively correlated with post-ischemiaangiogenesis. These ndings suggest that EE plays an important role in the recovery of damaged neuralReceived 12 June 2014Received in revised form 18 September 2014Accepted 13 October 2014Available online 20 October 2014

Keywords:Enriched environmentIschemiaNeural functionCD31AngiogenesisNeuroprotection, Fudan University, Shanghai 200032, China

a b s t r a c t

Increasing evidence shows that exposure to an enriched environment (EE) after cerebral ischemia/reperfusioninjury has neuroprotective benets in animal models, including enhancing functional recovery after ischemicstroke. However, the mechanism underlying this effect remains unclear. To clarify this critical issue, the currentstudy investigated the effects of EE on the improvement of damaged neural function and the induction ofangiogenesis. Adult rats were subjected to ischemia induced by middle cerebral artery occlusion followed byreperfusion. Neurological status scores were used to evaluate neural function on postoperative days 2, 7, and14. A beam-walking task was used to test the recovery of motor behavior on postoperative days 2, 5, 10, and15.We also used aMorriswatermaze task to examinewhether EE protected learning andmemory performance.The specic marker of angiogenesis of CD31 was examined by western blot. Angiogenesis around the peri-infarction regionwas assayed by laser scanning confocalmicroscopy (LSCM) after 14 days of EE exposure starting24 h after ischemia. Neurological status scores of animals in the EE groupwere signicantly higher than those inthe standard housing condition (SC) control group from the seventh day after ischemic. EE accelerated the recoveryof motor coordination and integration and also improved learning and memory performance after cerebrale Department of Rehabilitation Medicine, Jing'an District Centrf Department of Clinical Medicine, Shanghai Medical Collegee Hospital of Shanghai, 200040, Chinad Department of Sports Medicine and Rehabilitation, Shanghai Medical College, Fudan UniversEnriched environment induces angiogenesfunction outcomes in rat stroke model

Kewei Yu a,b, Yi Wu a,b,c,d,e,, Qi Zhang a,b, Hongyu Xie a

Shenyi Kuang f, Ruiping Hu a,d,a Department of Rehabilitation, Huashan Hospital, Fudan University, Shanghai 200040, Chinab State Key Laboratory of Medical Neurobiology, Fudan University, Shanghai 200032, Chinac The Yonghe Branch of Huashan Hospital, Fudan University, Shanghai 200436, Chinaon Medicine, Huashan Hospital,hai 200040, China. Fax: +86

hu79@163.com (R. Hu).and improves neural

Gang Liu a,c, Zhenzhen Guo a,b, Fang Li a,e, Jie Jia a,b,d,

ological Sciences

l sev ie r .com/ locate / jnsangiogenesis might play an important role in the recovery of the neuralfunction of stroke patients.

Thepresent study tested the hypothesis that EE can alleviate ischemicdamage and improve functional outcomes by increasing the emendationofmicrovascular structure and by inducing angiogenesis. Specically, we

investigated the effect of EE on behavioral status, angiogenesis, andexpression of CD31 after cerebral ischemic injury.

2. Material and methods

2.1. Animals

Healthy male SpragueDawley rats (Sino-British Sippr/BK LabAnimal Ltd., Shanghai, China) weighing 250280 g were housed at20 C on a 12 h light/dark cycle (lights on from 8:00 to 20:00) withfree access to water and standard rat chow. Animals were randomlyassigned to three experimental groups: ischemia with enriched envi-ronment (EE), ischemia with standard condition (SC), or sham surgerywith standard condition. Experiments were performed according to theGuidelines of the National Institutes of Health Guide for the Care andUse of Laboratory Animals. The protocol was approved by the Animal

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

2.4. Neural function test

2.4.1. Neurological status evaluationNeurological status was assessed on postoperative days 2, 7, and 14

in a blinded fashion using a seven-point scale [14] as follows: (0) nodecit; (1) failure to extend right forepaw fully; (2) decreased grip ofthe right forelimb when held by tail; (3) spontaneous movement in alldirections, but torso turning to right when held by tail; (4) circling orwalking to the right; (5)walking only in response to tactile stimulation;(6) no spontaneous activity; and (7) dead.

2.4.2. Beam-walking testWe used a beam-walking task to assess decits in coordination and

276 K. Yu et al. / Journal of the Neurological Sciences 347 (2014) 275280Experimental 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 proce-dure using a circulating heating pad. A 4-0 surgical nylonmonolamentwith a silicone tip was advanced from the external carotid artery intothe lumen of the internal carotid artery. After 90 min of cerebralischemia, the lament was withdrawn to allow reperfusion. In thesham-operated control group, rats underwent the same surgicalprocedure except for MCAO. Once animals recovered from anesthesia,they were scored based on a four-point scale [13] as follows: (0) noneurological symptoms; (1) unable to completely extend the frontcontralateral paw; (2) rotating while crawling and falling to the con-tralateral side; (3) unable towalkwithout assistance; and (4) uncon-sciousness [did not recover]. Rats with scores of 13 points wereconsidered successful models and were included in the study. Afterscoring, all rats were returned to SC home cages. After one day ofMCAO, 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 inenriched environment home cages (patent number of the People's Re-public of China: ZL 2010 2 0560281.3). As shown in Fig. 1, EE housingwas larger (80 cm wide 120 cm long 100 cm high) than standardhousing, and contained climbing platforms, plastic tubes and tunnels,chains, and small boxes. In addition, EE provided enhanced social stim-ulation because a total of 8 to 12 rats were group-housed, and animalswere changed every three days to maintain the novelty in the environ-ment. SC rats were group-housed in sets of three in standard cagesFig. 1. EE and SC houintegration of movement in the rats after ischemia, especially inhindlimbs [15]. The beam-walking apparatus consisted of a squarebeam (2.5 cm wide, 140 cm long, at a height of 42 cm) connected to ablack box (40 cm 20 cm 25 cm). The rats were trained for threedays to traverse the beam before ischemia induction and were testedfor beam-walk task performance the day before surgery to establish abaseline 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 asfollows: rat was not able to stay on the beam (0 points); rat did notmove but was able to stay on the beam (1 point); rat tried to traversethe beam but fell (2 points); rat traversed the beam with more than50% footslips of the affected hindlimb (3 points); rat traversed thebeam with more than one footslip but less than 50% (4 points); rathad only one slip of the hindlimb (5 points); and the rat traversed thebeam without any slips of the hindlimb (6 points) [16].

2.4.3. Morris water maze taskWe used the Morris water maze task to test the spatial learning and

memory of the rats as described previously [17]with somemodications.Briey, the water maze was a black circular pool lled with water(2123 C water temperature) and situated in a room with salientvisual cues. Spatial learning acquisition occurred during post-operative days 1619 for four consecutive days. Every rat received8 consecutive trials (with randomly assigned starting positions)per day to locate the platform that was submerged 2 cm beneaththe water surface. They were allowed 60 s to locate the platform,which was kept in a constant location. If they failed to locate the plat-form within 60 s, rats were manually guided to the platform and leftthere for 10 s. The mean escape latency per day was recorded for eachanimal and used in statistical analysis. One day after thenal acquisitiontraining session (day 20 post MCAO), all rats performed a probe testwith the escape platform removed. Animals were placed into the poolsing conditions.

in the location most distal to the target quadrant (with the platform re-moved). The percent of time spent in the target quadrant was recordedand 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 usedlectin, which selectively binds to endothelial glycocalyx only withinperfused vessels. Fluorescein isothiocyanate (FITC)-dextran (2 106molecular weight, Sigma; 1ml of 50 mg/ml) was administered intrave-nously to ischemic rats subjected to 15 days of tMCAO. This dye wasallowed to circulate for 1 min, after which the anesthetized animalswere killed by decapitation. Whole brains were quickly removed andplaced in 4% paraformaldehyde at 4 C for 48 h. Coronal sections(50 m thick) were cut on a vibratome for each rat; ten coronal sectionsof 1-mm intervals were obtained. The brain sections were analyzedwith a laser-scanning confocal imaging system. Eight brain regions in

3. Results

3.1. Neural function outcomes

3.1.1. Effect of EE on neurological statusSham rats had no neurological symptoms as indicated by neuro-

logical status scores of zero. As shown in Fig. 2A, therewas nodifferenceof neurological status between the EE group and the SC group onpostoperative day 2. However, EE rats showed signicant improvementrelative to SC rats (p b 0.05) on postoperative days 7 and 14.

3.1.2. Effect of EE on beam-walk taskAbeam-walk task assesses the decits in coordination and integration

of motor movement in rats after ischemia. As shown in Fig. 2B, ischemicrats with EE had better beam-walking performance as compared to SCrats (p b 0.05).

3.1.3. Effect of EE on spatial learning and memory

vemgm

277K. Yu et al. / Journal of the Neurological Sciences 347 (2014) 275280Fig. 2.Neural function outcomes. (A) Neurological status. EE rats showed signicant improative to SC controls, rats exposed to EEhad signicantly higher neurological scores, indicatin#All data are expressed as mean S.E.M. Statistical analyses wereperformed using SPSS 13.0 statistical software. We used one-wayANOVAs followed by LSD multiple comparison post hoc tests to be-tween groups. p values b 0.05 were considered to represent statisticallysignicant differences.Ipsilateral cortical tissue surrounding the ischemic zone was har-vested 15 days after MCAO. Cortical protein extracts and westernblotting analysis were performed as previously described [20]. Protein(40 g) was separated by 15% SDS-polyacrylamide gel electrophoresis(SDS-PAGE) and transferred to PVDFmembranes. The primary antibodywas anti-CD31 (Cell Signaling Technology). Anti-glyceraldehyde3-phosphate dehydrogenase (GAPDH: Cell Signaling Technology)was used as a loading control. Membranes were then washed and incu-bated with secondary antibodies for 1 h at room temperature. Quanti-cation of band intensity (optical density) was carried out on scannedwestern blot images using Image J software from blots of independentexperiments.

2.7. Statistical analysisthe ischemic boundary zone were selected within a reference coronalsection (interaural 8.8 mm, bregma 0.8 mm) [19]. Vascular surfacearea (mm2)was calculated by Image-Pro Plus software, and the numberof vascular branch points was counted in the microscope by a blindedinvestigator.

2.6. Western blottingp b 0.05 versus sham group.In addition to motor decits, cerebral ischemia is also known to im-pair spatial learning and memory depending on the location and sever-ity of the insult [21]. We examined whether EE protected learning andmemory performance following ischemic insult using the Morriswater maze. The initial assessment of spatial learning, as quantiedusing the metric of escape latency to nd the submerged platform, re-vealed that the three groups had no signicant difference on day 1(day 16 post tMCAO), but rats in the SC group took signicantly longerto nd the platform on days 2, 3, and 4 of training (days 17, 18, and 19post tMCAO, respectively), when compared to both the EE and shamgroups (Fig. 3A). There was no statistical difference between the EEand sham groups in spatial learning (Fig. 3A).

To evaluate spatial memory, we performed a probe trial on the dayafter the last escape latency trial (i.e., day 20 post tMCAO). During thistrial, the platformwas removed and the time rats spent in the quadrantof the former platform was measured (correct quadrant). The resultsindicated that rats in the SC group spent signicantly less time in thecorrect quadrant compared to both the EE and sham groups (Fig. 3B).There was no signicantly difference between the EE and sham group(Fig. 3B).

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

The neuroprotective effect provided by EE was suggestive of trueneurological rehabilitation, which is likely to be associated withangiogenesis. To test the hypothesis that post-stroke angiogenesisis enhanced by EE, we next examined whether EE affected revascu-larization in the ischemic boundary zone. As shown in Fig. 4, the

ents relative to SC rats on postoperative days 7 and 14. (B) Beam-walking test scores. Rel-otor function improvement. Data are expressed asmeanS.E.M. *p b 0.05 versus SC group,

vascular surface area and the number of vessel branch points werehigher in the EE group as compared to the SC group in the samevolume (p b 0.05).

3.3. Western blot analysis of CD31 expression

CD31 is the marker of angiogenesis. Therefore, we further assessedCD31 levels in the ischemic boundary zone by western blotting. Asshown in Fig. 5, western blot analysis revealed increased CD31 proteinexpression 15 days post MCAO in the EE group as compared to SCgroup (p b 0.05), indicating that EE indeed promoted angiogenesis in

using Spearman's correlation coefcients (r). Correlations betweenthe neurological function and angiogenesis are shown in Fig. 6. The neu-rological status scores were negatively correlated with angiogenesis(Fig. 6A, B) and cognitive functions were positively correlated withangiogenesis (Fig. 6C, D). These results imply that neural function out-comes are correlated with angiogenesis.

4. Discussion

The current study is the rst to investigate the effects of EE on angio-genesis and how angiogenesis is related to improved function outcomes

Fig. 3. Effect of EE on spatial learning andmemory. (A) Evaluation of spatial learning using theMorriswatermaze latency test following tMCAOdemonstrated that the three groups had nosignicant difference on day 16post tMCAO, but rats in the SC group requiremore time tond the platformondays 17, 18, and 19post tMCAO, respectively,when compared to both the EEand shamgroups. Therewas no signicant difference between the EE and shamgroups in spatial learning. (B) Determination of spatialmemory using the probe test onday 20post 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.

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

3.4. Correlation of neural function outcomes with angiogenesis

Finally, to assess a possible correlation between neurological functionoutcomes and angiogenesis, we compared the neurological status scoresand cognitive function scores to the quantied rate of angiogenesis byFig. 4. EE enhanced post-stroke revascularization in the ischemic boundary zone. The top panelbrains at 15 days after tMCAO. Bottom panels of the gure demonstrate how the vascular surfacpared to the SC group in the same volume. Data are expressed as mean S.E.M. *p b 0.05 versin an ischemic ratmodel. EE increased CD31 levels and promoted angio-genesis of rat cortex in an ischemic ratmodel. EE also accelerated the re-covery of coordination and integration of motor skills and improvedlearning and memory performance. Furthermore, we found that neuralfunction outcomes are signicantly correlatedwith angiogenesis. Takentogether, these results suggest that EE might promote angiogenesisafter ischemic injury, thus improving neural function outcomes.s show representative images of lectin uorescent signal in the ischemic boundary zone ofe area (A) and the number of vessel branch points (B) were higher in the EE group as com-us SC group, #p b 0.05 versus sham group.

EE is well established to protect against ischemia-induced brain inju-ry [22,2], especially in terms of improved function outcomes [23,24]. Pre-vious studies have suggested that EE is able to induce neuroplasticchanges following stroke, including neurogenesis [25,26], synaptogene-sis [27], increased dendritic branching and spine density [28,29] andneurotrophins [30,31]. All of these above changes may contribute tothe recovery of function outcomes after ischemic injury. Simultaneously,angiogenesis is essential for ischemic brain repair and function outcomerecovery because it stimulates blood ow and metabolism in the ische-mic boundary [32]. This is because the most important pathologicalevent that contributes to the impairment of brain function in the ische-mic brain is a shortage of glucose and oxygen supply. Neurons are highlysensitive to hypoxiaischemia. However, focal angiogenesismay providesufcient oxygen and nutrition for cerebral reconstruction by neuronalprogenitor cell differentiation and migration during the post-ischemia

period [33]. Therefore, an efcient vascular supply of blood is capableof preventing the progression of ischemic pathogenesis, including cere-bral infarction and tissue degeneration, andmay correlatewith function-al recovery after cerebral ischemia [34].

During the early acute phase of neurovascular injury, bloodbrainbarrier perturbations should predominate with key roles for variousmatrix proteases. During the delayed phase, brain angiogenesis mayprovide the critical neurovascular substrates for neuronal remodeling[35]. In our research, we focus on the long-term neurological functionrecovery, not the early acute phase of neurovascular injury. Togetherwith our results, EE improves neural function outcomes after cerebralischemia and promotes angiogenesis of the rat cortex in the ischemicboundary zone. Neural function outcomes are positively correlatedwith post-ischemia angiogenesis. Besides, one report has suggestedthat increased angiogenesis is not associated with increased cerebral

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. Opticaldensity 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.

279K. Yu et al. / Journal of the Neurological Sciences 347 (2014) 275280Fig. 6. Correlation of neural function outcomes with angiogenesis. r represents Spearman's correlation coefcient. p b 0.05 was considered a signicant correlation.

edema formation as well as no further destruction of the blood brain [10] Slevin M, Krupinski J, Slowik A, Kumar P, Szczudlik A, Gaffney J. Serial measurementof vascular endothelial growth factor and transforming growth factor-beta1 inserum of patients with acute ischemic stroke. Stroke 2000;31:186370.

280 K. Yu et al. / Journal of the Neurological Sciences 347 (2014) 275280tients with a higher density of blood vessels appear to have reducedmorbidity and survive longer [37]. Therefore, EE plays an importantand benecial role in the recovery of damaged neural function via regu-lation of angiogenesis after ischemia.

EE is a powerful tool to counteract cognitive and somatosensory def-icits and is a broad noninvasive strategy in treating neurological disor-ders following brain injury. At present, no studies have yet examinedthe effects of EE on angiogenesis after brain ischemia. CD31 (also calledPECAM-1) is expressed in all cells within the vascular compartment andplays an important role in angiogenesis [38]. CD31 is considered amarker of angiogenesis [39,40]. In our studies, we found that CD31 ex-pressionwas dramatically increased and that therewasmoremicrovas-culature in the peri-infarction region of the EE group as compared withthat in the SC group, suggesting that EE has can improve angiogenesisafter ischemia stroke. In addition, the neuron function outcome of theEE group was better than the SC group after MCAO. Furthermore, wefound neural function outcomes are correlated with the level of angio-genesis. Taking all of these observations into account, we speculatethat behavioral improvement after brain ischemia is due to angiogene-sis and that angiogenesis may be one of the recovery mechanisms thatEE induces following cerebral ischemia.

In conclusion, we have demonstrated that EE improves neural func-tion outcomes after cerebral ischemia and promotes angiogenesis of therat cortex in the ischemic boundary zone. Although these results are en-couraging in the study of stroke therapy, they still need to be conrmedon a larger scale and our future research program will elucidate themechanisms of EE-induced angiogenesis.

Conicts of interest

There are no conicts of interest.

Acknowledgments

The present study is supported by the National Natural ScienceFoundation of China (81171856, 81171855, 81401866), National Pro-gram on Key Basic Research Project of China (973 Program, no.2010CB945500) andMajor project of Shanghai Science and TechnologyCommission (no. 13411951000).

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Enriched environment induces angiogenesis and improves neural function outcomes in rat stroke model1. Introduction2. Material and methods2.1. Animals2.2. Middle cerebral artery occlusion (MCAO)2.3. Housing conditions2.4. Neural function test2.4.1. Neurological status evaluation2.4.2. Beam-walking test2.4.3. Morris water maze task

2.5. Vascular labeling and analysis of vascular density2.6. Western blotting2.7. Statistical analysis

3. Results3.1. Neural function outcomes3.1.1. Effect of EE on neurological status3.1.2. Effect of EE on beam-walk task3.1.3. Effect of EE on spatial learning and memory

3.2. EE enhances post-stroke revascularization in the ischemic boundary zone3.3. Western blot analysis of CD31 expression3.4. Correlation of neural function outcomes with angiogenesis

4. Discussion5. Conflicts of interestAcknowledgmentsReferences