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Research Report

Exogenous kallikrein enhances neurogenesis andangiogenesis in the subventricular zone and the peri-infarctionregion and improves neurological function after focal corticalinfarction in hypertensive rats

Li Ling, Qinghua Hou, Shihui Xing, Jian Yu, Zhong Pei, Jinsheng Zeng⁎

Department of Neurology and Stroke Center, the First Affiliated Hospital,Sun Yat-Sen University, No. 58 Zhongshan Road 2, Guangzhou 510080, P.R. China

A R T I C L E I N F O

⁎ Corresponding author. Fax: +86 20 87335935E-mail address: zengjs@pub.guangzhou.g

0006-8993/$ – see front matter © 2008 Elsevidoi:10.1016/j.brainres.2008.01.099

A B S T R A C T

Article history:Accepted 24 January 2008Available online 19 March 2008

Kallikrein, a serine proteinase, has been identified as an angiogenic growth factor recently.Weinvestigated whether delayed treatment with exogenous kallikrein enhances neurogenesisand angiogenesis after focal cortical infarction in stroke-prone renovascular hypertensive rats.Human tissue kallikrein (1.6×10−2 PNAU/kg) or vehicle was given through a tail vein daily for 6consecutive days starting 24 h after distal middle cerebral artery occlusion (MCAO). Cellproliferation was examined by using 5′-bromo-2′-deoxyuridine (BrdU, 50 mg/kg). Rats weresacrificed at 3, 7, 14 or 28 d after MCAO, respectively. Treatment with kallikrein significantlyincreased the number of BrdU+ cells in the subventricular zone (SVZ) and the peri-infarctionregion initiating 3 d after MCAO compared with the vehicle group (all p<0.05). Kallikreinsignificantly increased the number of BrdU+/DCX+ cells and BrdU+/nestin+ cells in the SVZ aswell as vascular density in the peri-infarction region compared with the vehicle group (allp<0.05), which increased at 3 d, peaked at 7–14 d after MCAO, and then gradually decreased.Kallikrein markedly increased the number of BrdU+/NeuN+ cells in the peri-infarction regioncomparedwith the vehicle group at 14 d and 28 d after MCAO (all p<0.05). The kallikrein groupshowed better functional improvement after stroke (all p<0.05). Our study demonstratesthat delayed administration of kallikrein at 24 h after cortical infarction promotes theSVZ neuroblasts proliferation, migration, and selective differentiation. Moreover, kallikreinenhanced endogenous neurogenesis is associated with angiogenesis, both attributing tofunctional improvement after stroke. Therefore, kallikrein may have a potential therapeuticperspective on ischemic stroke.

© 2008 Elsevier B.V. All rights reserved.

Keywords:KallikreinNeurogenesisAngiogenesisCerebral infarction

1. Introduction

It is well known that endogenous neurogenesis existsmainly intwo regions of the adult brain: the subventricular zone (SVZ) ofthe lateral ventricle and the subgranular zone (SGZ) of the

.d.cn (J. Zeng).

er B.V. All rights reserved

hippocampal dentate gyrus (Lois and Alvarez-Buylla, 1993;Doetsch andAlvarez-Buylla, 1996; Eriksson et al., 1998). Cerebralischemia can stimulate endogenous neurogenesis in the SVZand the SGZ (Liu et al., 1998; Zhang et al., 2001; Jin et al., 2001;Ohab et al., 2006). Angiogenesis is an important process for

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forming new brain microvessels after cerebral ischemia, whichimproves tissue microperfusion around the ischemic boundaryregion. Recently, substantial studies have documented thatneurotrophic andgrowth factors canpromoteneurogenesis andangiogenesis, as well as improve neurological function aftercerebral ischemia (Jin et al., 2002; Wang et al., 2004; Chen et al.,2003, 2004; Shyu et al., 2004; Sugiura et al., 2005; Ardelt et al.,2007; Sehara et al., 2007). Furthermore, endogenous neurogen-esis is coupled with angiogenesis in a neurovascular niche, amicroenvironment in which newly born neuroblasts are as-sociated with vascular endothelial cells after ischemic stroke(Zhang et al., 2005; Yamashita et al., 2006; Ohab et al., 2006).

Tissue kallikrein, a glycoprotein of the serine proteinasesuperfamily, cleaves lowmolecular weight kininogen to releasevasoactive kinins. Kinins in turn bind to high-affinity bradykininB1 or B2 receptors, then triggering a series of biological effects(Emanueli and Madeddu, 2003; Chao and Chao, 2004). Recently,kallikrein has been identified as a novel angiogenic growthfactor, which is effective and durable even at a small dose due toits enzymatic nature (Emanueli and Madeddu, 2003, 2004). Allthe components of the kallikrein/kinin system are expressed inbrain (Walker et al., 1995) and up-regulated by ischemic stroke(Wagner et al., 2002; Groger et al., 2005). However, only a fewstudies have investigated the potential effects of kallikrein oncerebral ischemia (Xia et al., 2004, 2006). A recent study hasshown that tissue kallikrein gene transfer protected againstcerebral ischemia by promoting glial cell survival andmigrationand inhibiting apoptosis (Xia et al., 2004). Moreover, treatmentwith kallikrein gene increased proliferative neurons and capil-lary density in the ischemic penumbra after stroke, suggesting

Fig. 1 – Exogenous kallikrein treatment enhances BrdU+/DCX+ dsubventricular zone (SVZ). Administration of exogenous kallikrein the dorsolateral ventricle wall (A–C) and in the lateral ventricland J–L) at 7 d after middle cerebral artery occlusion (MCAO). QuanSVZ at each time point for each group. *p<0.05 compared with th

that the neuroprotective action of kallikreinmight be associatedwith newly born neurons and vessels (Xia et al., 2006). However,the detailed information on how kallikrein affects neurogenesisand angiogenesis after stroke, for example, the source of neuralstem cells proliferation and migration path has not been in-vestigated. It is also unknown whether there is a relationshipbetween thenewlybornneuroblasts and vessels after treatmentwith kallikrein. Additionally, compared with adenovirus-me-diated gene transfer, exogenous kallikrein causes fewer sideeffects, thus having more clinical application perspectives (Em-anueli and Madeddu, 2003). The present study was designed toinvestigate in detail the potential effects of exogenous humantissue kallikrein on neurogenesis and angiogenesis after focalcortical infarction in hypertensive rats with a focus on cellproliferation, migration, and differentiation. Furthermore, thepotential role of kallikrein on interaction between poststrokeneurogenesis and angiogenesis was also examined.

2. Results

2.1. Blood pressure

The kallikrein group did not show any alteration in bloodpressure at 7 d after middle cerebral artery occlusion (MCAO)compared with that before MCAO (213±13.5 mmHg vs 209±15.2 mmHg, pN0.05). There was no significant difference inblood pressure between the kallikrein group and the vehiclegroup at 7 d after MCAO (213±13.5 mmHg vs 216±16.4 mmHg,pN0.05).

ouble-labeled neural stem cells proliferation in thein increased the number of BrdU+, DCX+ and BrdU+/DCX+ cellse wall (D–F) of SVZ compared with the vehicle group (G–I,titative data of BrdU+ cells (M) and BrdU+/DCX+ cells (N) in thee vehicle group. Bar=100 μm.

Fig. 2 – Exogenous kallikrein treatment enhances BrdU+/nestin+ double-labeled neural stem cells proliferation in the SVZ.Administration of exogenous kallikrein increased BrdU+, nestin+ and BrdU+/nestin+ cells in the dorsolateral ventricle wall(A–C) and in the lateral ventriclewall (D–F) of SVZ comparedwith the vehicle group (G–I, and J–L) at 7 d afterMCAO.Quantitativedata of BrdU+/nestin+ cells in the SVZ at each time point for each group (M). *p<0.05 compared with the vehicle group.Bar=100 μm.

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2.2. Infarction volume and Neurological evaluation

Hematoxylin and eosin (H&E) staining showed that the cor-tical infarction was confined to the right temporoparietalcortex, and was similar in size in all ischemic animals. Therewas no significant difference in infarction volume betweenthe kallikrein group and the vehicle group at all time pointsover observation period (23.6±1.85% vs 24.9±1.77% at 3 d,19.1±2.93% vs 20.7±2.75% at 7 d, 16.2±1.62% vs 17.2±1.55%at 14 d, 10.9±1.38% vs 12.0±1.96% at 28 d after MCAO, re-spectively, all pN0.05). However, compared with the vehicletreated rats, the kallikrein treated rats exhibited better neu-rological scores at 3, 7, 14 and 28 d after onset of ischemia (2(1.25, 2) vs 3 (2.25, 3) at 3 d, 1 (1, 1.75) vs 2 (2, 2.75) at 7 d, 0.5 (0,1) vs 1.5 (1, 2) at 14 d, 0 (0, 0.75) vs 1 (1, 1) at 28 d after MCAO,respectively, all p<0.05).

Fig. 3 – Exogenous kallikrein enhances the migration of newly gcortex. Representative double immunofluorescent staining show(C, arrow shows), labeled by BrdU+/DCX+, migrate from the SVZ toMCAO in the kallikrein group. Bar=100 μm.

2.3. Cell proliferation in the SVZ and the peri-infarctionregion

5′-bromo-2′-deoxyuridine (BrdU)-positive cells were detectedin the bilateral SVZ after MCAO. In ischemic animals, thenumber of BrdU-positive cells was higher in the ipsilateral SVZthan that in the contralateral SVZ. In addition, the ischemiainduced BrdU-positive cells were mainly located in the dor-solateral ventricle wall (Figs. 1B and H) and also in the lateralventricle wall to a lesser degree (Figs. 1E and K). Besides, BrdU-positive cells were increased in the corpus callosum andstriatum ipsilateral to MCAO compared with the contralateralside (data not shown). Administration of kallikrein signifi-cantly increased the number of BrdU-positive cells in theipsilateral SVZ (Figs. 1B, E, H, K and M, all p<0.05) and in theperi-infarction cortex (321±33.4 vs 215±33.6 at 3 d, 490±82.0 vs308±51.5 at 7 d, 295±65.1 vs 191±65.7 at 14 d, 137±16.2 vs 109±

enerated neural stem cells from the SVZ to the peri-infarctions that neural stem cells with elongated nuclei and biolarthe peri-infarction cortex in a chain-like structure at 7 d after

Fig. 4 – Exogenous kallikrein enhances the differentiation of newly generated neural stem cells into mature neurons.Representative double immunofluorescent staining shows increased BrdU+/NeuN+ cells in the peri-infarction cortex in thekallikrein group (C) compared with the vehicle-treated group (F) at 14 d after MCAO. *p<0.05 compared with the vehicle group.Bar=40 μm.

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15.9 at 28 d after MCAO, respectively, all p<0.05) comparedwith the vehicle group. The number of BrdU-positive cellsincreased at 3 d after MCAO, peaked at 7–14 d and graduallydeclined thereafter, but a few were still present at 28 d afterMCAO. These data indicate that administration of exoge-nous kallikrein enhances cell proliferation after focal corticalinfarction.

2.4. Proliferation, migration, and differentiation ofendogenous neural stem cells

DCX, a microtubule-associated protein, is a marker of migra-tory or immature neurons, and nestin, an intermediate fila-

Fig. 5 – Exogenous kallikrein enhances angiogenesis in the periin the peri-infarction cortex in the kallikrein group (A) compareddata of vascular density in the peri-infarction cortex at each timegroup. Bar=200 μm.

ment protein, is a neuroepithelial stem cellmarker. Treatmentwith kallikrein significantly increased the number of bothBrdU+/DCX+ cells (Figs. 1C, F and N, all p<0.05) and BrdU+/nestin+ cells (Figs. 2C, F, and M, all p<0.05) in the ipsilateralSVZ compared with vehicle group (Figs. 1I, L and N, and 2I, L,and M) at all time points after MCAO. The dynamic changes innumber of newly generated neural stem cells were in parallelwith that of BrdU-positive cells, that is, newly generatedneural stem cells also increased at 3 d after MCAO, peaked at7–14 d, then gradually decreased, nearly disappeared 28 d afterMCAO. Furthermore, BrdU+/DCX+ cells appeared to migratefrom the SVZ to the peri-infarction cortex in a chain-likestructure, which were more obvious in the kallikrein treated

-infarction cortex. Representative images of vascular densitywith the vehicle group (B) at 7 d after MCAO. Quantitativepoint for each group (C). *p<0.05 compared with the vehicle

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rats (Fig. 3). These results suggest that exogenous kallikreinmay promote proliferation of neuroblasts in the SVZ and mi-gration of neuroblasts from the SVZ to the peri-infarction cor-tex after focal cortical infarction.

To further investigate whether the proliferative andmigra-tory neural stem cells can differentiate into functional cells,double immunofluorescent labeling was performed using dif-ferent cell type-specific markers (NeuN, a marker of matureneurons; GFAP, a marker of astrocytes) and BrdU. Treatmentwith kallikrein significantly increased the number of BrdU+/NeuN+ cells (21±3.4 vs 13±2.6 at 14 d, 16±3.2 vs 10±2.8 at 28 dafter MCAO, respectively, all p<0.05) in the peri-infarctioncortex after MCAO compared with the vehicle group (Figs. 4Cand F). However, no BrdU+/GFAP+ cell was found in the peri-infarction cortex either in the kallikrein group or in the vehiclegroup after MCAO (data not shown). These data indicate thatexogenous kallikrein may enhance the selective differentia-tion of newly generated neural stem cells intomature neuronswithin the peri-infarction region after cortical infarction.

2.5. Angiogenesis in the peri-infarction region and therelationship between neurogenesis and angiogenesis

Laminin is a basal membrane marker of blood vessel. Ad-ministration of kallikrein significantly increased vasculardensity in the peri-infarction cortex compared with the ve-hicle group (Figs. 5A–C, all p<0.05). Blood vessels withenlarged walls and vessels with some BrdU-labeled nucleion the endothelial cells were detected within the boundary ofischemic cortex in the kallikrein treated rats. These datasuggest that exogenous kallikreinmay enhance angiogenesisafter focal cortical infarction.

Newly born vascular endothelial cells which were labeledwith BrdU+/laminin+ were clearly observed in both the lateraland the dorsolateral SVZ ipsilateral toMCAO (Fig. 6), where theendogenous neural stem cells proliferate and migrate from,indicating that endogenous neurogenesis may be associatedwith angiogenesis in kallikrein treated rats after focal corticalinfarction.

Fig. 6 – Angiogenesis is associated with endogenous neurogenedouble immunofluorescent staining of BrdU+/laminin+ cells (arroventricle wall (F) of the SVZ at 7 d after MCAO. Bar=100 μm.

3. Discussion

In the present study, we found that treatment with exogenouskallikrein beginning 24 h after stroke significantly increasedendogenous neural stem cell proliferation, migration and dif-ferentiation. Besides, exogenous kallikrein also boosted post-stroke angiogenesis, which was associatedwith neurogenesis.Our data suggest that exogenous kallikrein enhanced neuro-genesis and angiogenesis may contribute to functional im-provement after stroke.

In this study, we used stroke-prone renovascular hyper-tensive rats (RHRSP) to create a focal cortical infarctionmodel.This model has several advantages to investigate neurogen-esis after stroke. First, the focal cortical infarction model issuitable to study the newly formed neural stem cellsmigratingfrom the SVZ to the peri-infarction cortex in a long distancebecause the ischemic lesion restricted to the temporoparietalcortex in rats of distalMCAO (Ohab et al., 2006). In addition, thelocalization and size of cortical infarction are highly repro-ducible in this model of distal MCAO because the intracranialsmall arteries and arterioles are impaired in RHRSP (Zeng andHuang, 1998). Furthermore, hypertensive rats are better tomimic clinical practice, in which hypertension is one of themost important risk factors for ischemic stroke (Gillum andSempos, 1997).

Previously, the beneficial effects of kallikrein gene transferhave been documented in an animal model of ischemia-reperfusion-induced cerebral infarction. Similarly, we furtherdemonstrated that treatment with exogenous kallikrein sig-nificantly improved neurological function in a distal MCAOmodel. The improvement continued over all observation timepoints from 3 d to 28 d after stroke, indicating that exogenouskallikrein has long lasting beneficial effects on ischemic brainand a short period of administration of exogenous kallikreincan persistently improve neurological function over a relativelong period time. However, treatment with exogenous kallik-rein did not reduce infarction volume after stroke, which isinconsistent with previous studies showing that kallikrein

sis in exogenous kallikrein treated group. Representativew) in the lateral ventricle wall (C) and in the dorsolateral

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gene transfer reduced infarction volume (Xia et al., 2004, 2006).Thediscrepancymay be explained by thedifferences in animalmodels, therapy, and time of administration. It is reasonablethat infarcts were formed and irreversible when we initiatedtreatment at 24 h after MCAO. Treatment of stroke with seve-ral therapeutic agentshas been shown to improveneurologicalfunction without reducing infarction volume (Wang et al.,2004; Li et al., 2004; Ardelt et al., 2007). The protectivemechanisms of these agents are partially associated withpoststroke neurogenesis and angiogenesis. Nevertheless, thedata here documented that exogenous kallikrein significantlyimproved neurological function even when it started at 24 hafter stroke.

Consistent with previous reports, we found that cerebralischemia induced BrdU-positive cells in the SVZ and the peri-infraction cortex. Treatment with exogenous kallikrein sig-nificantly increased the number of BrdU-positive cells in theSVZ and the peri-infarction cortex, indicating that kallikreinpromotes cell proliferation after stroke. The majority of pro-liferative cells in the SVZ are likely to be endogenous neuralstem cells because the number of both BrdU+/DCX+ cells andBrdU+/nestin+ cells was increased in the SVZ in kallikreintreated rats, which was in parallel with the increase of BrdU-labeled cells. Although it has been reported that kallikreingene transfer increases proliferative neurons in the ischemicpenumbra in vivo and kinin stimulates neuronal proliferationin primary cultured neuronal cells in vitro (Xia et al., 2006), thesource of the newly born neuroblasts remains unknown. Wefound that DCX+ cells and nestin+ cells persistently increasedin ventricle wall after stroke. Anatomically, DCX+ cells werelargely located in the dorsolateral ventricle wall while nestin+

cells predominantly in the lateral ventricle wall. Moreover,BrdU+/DCX+ cells in the dorsolateral ventricle wall displayed atypical morphology of migrating neuroblasts with elongatednuclei and biolar, and migrated from the SVZ to the peri-infarction cortex in a chain-like structure. These results in-dicate that exogenous kallikrein not only enhances prolifera-tion of neural stem cells in the SVZ, but also promotes themigration of newly generated neural stem cells from the SVZto the ischemic region, which may contribute to repair is-chemic injury.

Function is the key principle for determiningwhether adultneurogenesis is successful. Neuroblasts have no impact on thebrain function without differentiating into mature neuronsand integrating into the parenchymal tissue. Using co-labelingof BrdU with a mature neuronal marker NeuN+, we investi-gated whether exogenous kallikrein can enhance differentia-tion of immature neurons intomature neurons.We found thatexogenous kallikrein significantly increased the number ofBrdU+/NeuN+ cells in the peri-infarction cortex, which is con-sistent with a previous study (Xia et al., 2006). No BrdU+/GFAP+

cell was detected in the peri-infarction region in either thekallikrein group or the vehicle group. These results indicatethat exogenous kallikrein may promote selective differentia-tion of the neural stem cells intomature neurons in themodelof focal cortical infarction.

As a novel angiogenic factor, kallikrein gene has been suc-cessfully applied in therapeutic angiogenesis in various expe-rimental ischemicmodels, such as, hindlimb ischemia, cardiacinfarction and renal ischemia (Porcu et al., 2002; Emanueli

et al., 2000, 2001a,b; Yoshida et al., 2000; Agata et al., 2002).Recently, kallikrein gene transfer has been shown to promoteangiogenesis after ischemia-reperfusion-induced cerebralinfarction (Xia et al., 2006). Several mechanisms are involvedin kallikrein/kinin systems-enhanced angiogenesis, such asstimulating endothelial cell proliferation, increasing NO forma-tion through eNOS activation, reducing oxidative stress, inhibit-ing apoptosis and inflammation and increasing cAMP as well ascGMP levels (Emanueli et al., 2001a,b; Emanuelia and Madeddu,2003). We found that treatment with exogenous kallikreinsignificantly increased not only vascular density but alsoBrdU+/laminin+ cells in the peri-infarction cortex. Besides,kallikrein also increased vascular density in other regions suchas the corpus callosum, the striatum, even the SVZ in theipsilateral side. These results indicate that exogenous kallikreinenhances angiogenesis after cortical infarction. Poststrokeangiogenesis has been documented to be coupled with neuro-genesis in a neurovascular niche after cerebral ischemia (Zhanget al., 2005; Ohab et al., 2006; Yamashita et al., 2006). Newlyformed endothelial cells, produce various vascular growthfactors and neurotrophic factors such as brain-derived neuro-trophic factor, vascular endothelial growth factor and basicfibroblast growth factor. Consequently, these neurotrophicfactors foster neural stem cell survival (Chen et al., 2003; Wanget al., 2004), which in turn improves neurological function.Therefore,we testedwhether exogenouskallikreinhaspotentialeffect on the relationship between neurogenesis and angiogen-esis after cerebral ischemia. We indeed detected some BrdU+/laminin+ cells within the SVZ “neurovascular niche” regionwhere neuroblasts generate, andwithin the chain-like structurefor neuroblasts migration from the SVZ to the peri-infarctioncortex (Zhang et al., 2005; Yamashita et al., 2006; Ohab et al;2006). These results indicate that stroke-induced endogenousneurogenesis may be associated with angiogenesis after treat-ment with kallikrein, and these two regenerative elementsmaycontribute to the neurological functional recovery after stroke.

It is still controversial whether human tissue kallikreinreduces blood pressure in hypertensive animals (Chao et al.,1998; Wolf et al., 2000; Emanueli et al., 2001b). In this study,we did not detect any alteration of blood pressure in exo-genous kallikrein treated renovascular hypertensive rats.

In summary, our findings suggest that the beneficial effectsof exogenous kallikrein on neurological function may bemediated by at least two different mechanisms: neurogenesisand angiogenesis. The present study provides novel evidencesthat human tissue kallikrein may have a potential therapeuticperspective on ischemic stroke.

4. Experimental procedures

4.1. Animals and focal cortical infarction model

All experimental procedures were approved by local Institu-tional Animal Case and Use Committee and in accordancewith the guidelines for animal use. The most effort was madeto minimize the animals' suffering during the experiment.

Firstly, RHRSP were induced by our previously describedmethod (Zeng et al., 1998). In brief, a total of 48 male Sprague–Dawley rats weighing 80–100 g were anesthetized with 10%

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chloral hydrate (3 ml/kg, intraperitoneally), then underwentan operation of renal artery constriction with two-kidney twoclip methods. Systolic blood pressure was measured by a tail-cuff sphygmomanometer in preheated (37 °C, 15 min) con-scious rats atbaselineandweeklyafter renal artery constriction.Secondly, twelve weeks later, the rats weighing 300–400 g,whose systolic blood pressure was steady and higher than180 mmHg but without stroke symptoms, were subjected toproduce focal cortical infarction using a previously describedmodel of distal MCAO (Bederson et al., 1986). Briefly, ratswere anesthetized as the above method. The right middlecerebral artery was exposed and then occluded above therhinal fissure and distal to the striate branches by bipolarcoagulation under a surgical microscope. Body temperaturewas maintained at 37±0.5 °C during the surgical and re-covery period using a heat lamp. The rats were sacrificed(n=6 for each group at each time point) at 3, 7, 14 or 28 d afterMCAO respectively.

4.2. Human tissue kallikrein administration and BrdUlabeling

The MCAO rats were randomly assigned to 2 groups. Twenty-four rats in the kallikrein group were injected with 1.6×10−2 PNAU/kg human tissue kallikrein (Techpool Bio-PharmaCo., Ltd, Guangdong, China, certification: H20052065) through atail vein daily for 6 consecutive days starting 24 h after MCAO,while 24 rats in the vehicle group were injected with the samevolume of 0.9% saline at the same time. BrdU (50mg/kg, Sigma-Aldrich, USA), a thymidine analog that is incorporated into theDNA of dividing cells during S-phase, was used to label pro-liferative cells. BrdU was injected intraperitoneally twice dailyfor 6 consecutive days starting 24 h after MCAO in all rats.

4.3. Blood pressure measurement

To monitor the possible side effect of exogenous kallikrein ondecreasing blood pressure, systolic blood pressure was mea-sured by a tail-cuff sphygmomanometer in preheated (37 °C,15 min) conscious rats before and at 7 d after MCAO in all ex-perimental rats.

4.4. Neurological functional assessment

To evaluate neurological function, Bederson scores (Bedersonet al., 1986) were assessed as previously described, at 3, 7, 14 or28 d after MCAO by an investigator who was blinded to the ex-perimental groups. The rat was suspendedwith its tail at about20 cm above the floor. The normal response with extension ofboth forelimbs toward the floor was scored 0. Flexion of the leftlimb toward the body and/or rotation of the left shoulder andlimb medially were regarded as abnormal posture. When theabnormal posture was observed, the rat would be placed on asheet of soft plastic-backed paper that could be gripped by itsclaws. Lateral pressurewould be applied frombehind the shoul-ders to slid the forelimbs gently to the left and then to the right.Resistance to sliding inbothdirectionswas scored1,adecreasedresistance to the lateral push from the right side was scored 2,and spontaneous anti-clockwise circling or left-sided tumblingor unmoving was scored 3.

4.5. Tissue preparation

Rats were anesthetized with 10% chloral hydrate (3 ml/kg,intraperitoneally) and sacrificed by transcardiac perfusionwith heparinized saline followed by 4% paraformaldehyde in0.1 M phosphate buffer (PB, pH=7.4). After postfixation for 6 h,the brains were immersed in 15%, 20%, 30% sucrose/PBsolution sequentially until they sank, then 10 μm coronal sec-tions were cut on a cryostat vibratome (VT 2800 N; Leica,Heidelberg, Germany).

4.6. Infarction volume measurement

Wemeasured relative infarction volume at 3, 7, 14 or 28 d afterMCAO using H&E staining, as described previously (Swansonet al., 1990). Relative infarction volume was expressed as thepercentage of the contralateral hemisphere and analyzed bythe NIH Image J 1.38 program.

4.7. Immunohistochemistry

For single- or double-label immunofluorescent staining, thefollowing primary antibodies were used in this study: mousemonoclonal anti-BrdU (1:1000, Sigma-Aldrich, USA), sheeppoly-clonal anti-BrdU (1:500, Abcam, USA), goat polyclonal anti-doublecortin (DCX, 1:200, Santa Cruz, USA), mouse monoclonalanti-nestin (1:1000, Chemicon, USA), mouse monoclonal anti-neuronal nuclei (NeuN, 1:1000, Chemicon), rabbit polyclonalanti-glial fibrillary acidic protein (GFAP, 1:1000, GeneTex, USA),rabbit polyclonal anti-laminin (1:200, Sigma-Aldrich). Afterbeing incubated with primary antibodies at 4 °C overnight, thefluorescent-labeled secondary antibodiesAlexa Flour 488 (1:200,Invitrogen, USA), Cy3-conjugated antibody (1:200, Chemicon),Cy2 or Cy3-conjugated antibody (1:200, Jackson Immunore-search Laboratories, USA) were applied for 1 h at room tem-perature. For BrdU immunofluorescence, brain sections werepretreated with 2 N HCl at 37 °C for 30 min, and then rinsed in0.1M boric acid (pH=8.5) at room temperature for 10min beforebeing incubated with blocking solution. Negative control sec-tions were incubated with 0.01 M phosphate buffer saline as asubstitute for the primary antibody. The images were capturedwith a microscope (Olympus BX51, Japan).

4.8. Cell counting

BrdU- or double-immunopositive cells were counted blindlyin six 10 μmcoronal sections per animal, spaced 200 μmapart.Cells were counted under high-power (×40 objective) on amicroscope (Olympus BX51, Japan) with a Magnifire digitalcamera, and the image was analyzed by the NIH Image J 1.38program. Results were expressed as the average number ofBrdU or double-positive cells per section.

For measurement of vascular density, images of the peri-infarctioncortexweredigitizedwithin the field (850μm×640μm)under a ×20 objective via the NIH Image J 1.38 program. Datawere expressed as laminin-immunopositive area percentage.

4.9. Statistical analysis

Numerical data were presented as means±SEM. All numericaldata were analyzed with SPSS 13.0 (SPSS Inc., Chicago, IL,

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USA). The comparisons of different groups were analyzed bythe two-tailed Student's t test or Mann–Whitney U test. Ap value less than 0.05 was considered statistically significant.

Acknowledgments

This studywas supported by the grants from the Teaching andResearch Award Program for Outstanding Young Teachers inHigher Education Institutions of the Ministry of Education,China (2002), the National Natural Science Foundation ofChina (Nos. 39940012, 30271485 and 30770764), the NaturalScience Foundation of Guangdong Province, China (Nos.990065, 21906, and 2003C30610), China Medical Board of NewYork Inc. (No. CMB00-730), the Fund for Priority Subjects inClinical Medicine, Chinese Ministry of Health (2004), the Keyand Scientific Project of the Natural Science Foundation ofGuangdong Province, China (Nos. 2003B30303, 2003C30610 and2003D30301), the Natural Science Foundation for DoctorialResearch of Guangdong Province (No.5300761) and the fund oncollaboration study for First Affiliated Hospital and Life Sci-ence Institute in Sun Yet-Sen University (2006), the Founda-tion for Scientific and Technologic Project of Guangzhou City(Nos. 2006 Z1-E0111 and Z1-E0113).

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