Transplanted bone marrow stromal cells protect neurovascular units and ameliorate brain damage in stroke-prone spontaneously hypertensive rats

Original Article neup_1291 1..12

Transplanted bone marrow stromal cells protectneurovascular units and ameliorate brain damagein stroke-prone spontaneously hypertensive rats

Masaki Ito, Satoshi Kuroda, Taku Sugiyama, Katsuhiko Maruichi, Masahito Kawabori,Naoki Nakayama, Kiyohiro Houkin and Yoshinobu Iwasaki

Department of Neurosurgery, Hokkaido University Graduate School of Medicine, Sapporo, Japan

This study was aimed to assess whether bone marrowstromal cells (BMSC) could ameliorate brain damagewhen transplanted into the brain of stroke-prone sponta-neously hypertensive rats (SHR-SP). The BMSC or vehiclewas stereotactically engrafted into the striatum of maleSHR-SP at 8 weeks of age. Daily loading with 0.5% NaCl-containing water was started from 9 weeks. MRIs and his-tological analysis were performed at 11 and 12 weeks,respectively. Wistar-Kyoto rats were employed as thecontrol. As a result, T2-weighted images demonstratedneither cerebral infarct nor intracerebral hemorrhage, butidentified abnormal dilatation of the lateral ventricles inSHR-SP. HE staining demonstrated selective neuronalinjury in their neocortices. Double fluorescence immuno-histochemistry revealed that they had a decreased densityof the collagen IV-positive microvessels and a decreasednumber of the microvessels with normal integrity betweenbasement membrane and astrocyte end-feet. BMSC trans-plantation significantly ameliorated the ventricular dilata-tion and the breakdown of neurovascular integrity. Thesefindings strongly suggest that long-lasting hypertensionmay primarily damage neurovascular integrity andneurons, leading to tissue atrophy and ventricular dilata-tion prior to the occurrence of cerebral stroke. The BMSCmay ameliorate these damaging processes when directlytransplanted into the brain, opening the possibility of

prophylactic medicine to prevent microvascular andparenchymal-damaging processes in hypertensive patientsat higher risk for cerebral stroke.

Key words: bone marrow stromal cell, cell transplantation,neurovascular unit, neurovascular protection, stroke pronespontaneously hypertensive rat.

INTRODUCTION

Currently, it is quite difficult to restore lost neurologicalfunctions after cerebral stroke. A growing number ofstudies have highlighted the potential of bone marrowstromal cell (BMSC) transplantation as a novel therapeuticapproach for cerebral stroke, and multiple mechanismshave been demonstrated through which BMSC protect andrepair the damaged CNS, including after stroke.1,2 Thus,BMSC could survive, extensively proliferate and migratewhen stereotactically transplanted into the infarct brainof rodents.3–7 First, cultured BMSC have the potential todifferentiate into neural cells and replace injured CNStissue.8–14 Second, transplanted BMSC may enhance endog-enous repair mechanisms by increasing synaptophysinexpression in the penumbra,15 or by enhancing axonalregeneration or axonal sprouting to restore connectionsbetween different cerebral areas.16,17 Third, they maypromote angiogenesis in damaged CNS tissue by secretingangiogenic factors such as VEGF.18,19 Finally, they mayenhance the survival of the host neurons and/or enhancean endogenous neurogenesis by creating a nursingenvironment.20–23

On the other hand, stroke-prone spontaneously hyper-tensive rats (SHR-SP) were produced in 1974 by Okamotoet al. by proper selective breeding of spontaneously hyper-tensive rats (SHR). Thus, successive selection of SHR ofwhich one or both parents developed cerebral stroke

Correspondence: Satoshi Kuroda, MD, PhD, Department of Neuro-surgery, Hokkaido University Graduate School of Medicine, North 15West 7, Kita-ku, Sapporo, Hokkaido 060-8638, Japan. Email: [email protected]

Disclosure of finding: This study was supported by Grant-in-aidfrom the Ministry of Education, Science and Culture of Japan(No.20591701, No.20390377, No.21390400, and No.23390342).

Received 28 October 2011; revised and accepted 11 December2011.

Neuropathology 2012; ••, ••–•• doi:10.1111/j.1440-1789.2011.01291.x

© 2012 Japanese Society of Neuropathology

spontaneously, greatly increased the spontaneous inci-dence of cerebral lesions such as cerebral infarct, intrac-erebral hemorrhage and white matter rarefaction up toabout 80% in males over 100 days of age.24 Moreover, whenfed by a high-salt diet, disease development was acceler-ated by raising peripheral resistance and increasing bloodpressure.24,25 More importantly, histological studies showeda vessel-based pathologic process with several features,including atherosclerotic plaque, medial degeneration,fibrinoid necrosis, hypertrophy and thrombosis. These find-ings are very similar to those in patients with cerebralstroke.26–32 Using this animal model of stroke, numeroushistological studies and intervention studies have been per-formed.33 However, there are no studies that denote thetherapeutic effects of cell transplantation therapy on theoutcome in SHR-SP.

Based on these observations, this study was aimed toevaluate whether BMSC transplantation could prevent ordelay the pathologic processes in cerebral vasculaturesand brain in SHR-SP. For this purpose, BMSC were ste-reotactically transplanted into the brain of 8-week-oldSHR-SP. Pathologic processes in the brain were moni-tored, using MRI and histological analysis. As recentlypointed out, a general assessment of the causes of thefailure of neuroprotectants have led to a shift in perspec-tive from a focus on the neurons alone to a focus on thecomplex of neurons, the microvessels that supply themand the supportive cells (astrocytes, other glial cells andresident inflammatory cells). Neurovascular units (NVU)have been proposed as a conceptual framework.34 There-fore, the integrity of NVU was precisely analyzed in thisstudy, using double fluorescence immunohistochemistry.

MATERIALS AND METHODS

Animals and study design

All animal experiments were approved by the AnimalStudies Ethical Committee at Hokkaido UniversityGraduate School of Medicine. Six-week-old maleSHR-SP (n = 7) were purchased from Japan SLC (Shi-zuoka, Japan). As the control, we used 6-week-old maleWistar Kyoto rats (WKY, n = 3, Japan SLC) that are nor-motensive, but are a closely related strain of SHR-SP. Allanimals were fed by stroke permissive diet, including20.8% proteins, 0.74% potassium and 0.4% sodium(Funahashi Farm Co. Ltd., Chiba, Japan). They wereweighed, and their blood pressure was measured by thetail-cuff method (BP-98 A, Softron, Tokyo, Japan) everyweek. At the age of 8 weeks, BMSC or vehicle was ster-eotactically transplanted into the brain of SHR-SP (n = 3and 4, respectively). From 9 weeks of age, 0.5% NaCl was

loaded every day in the drinking water. The precise meth-odology is described below.

Isolation, culture and stereotactic transplantationof rat BMSC

Bone marrow stromal cells were harvested under sterileconditions from 8-week-old female SHR-SP, as describedpreviously.4,35 Briefly, the femurs were dissected and bothends were cut. Then, the marrow was extruded with 5 mLof DMEM (Sigma-Aldrich, Tokyo, Japan) containing10% fetal bovine serum (FBS), 2 mmol/L L-glutamine,100 units/mL penicillin G, and 10% heparin, using a 2.5-mLsyringe and a 21-gauge needle. Between 10 and 15 ¥ 106

whole marrow cells were plated in a 75-cm2 tissue cultureflask that was coated with collagen I (Becton DickinsonLabware, Lawrence, KA, US), in DMEM/10% FBS. Cellswere incubated at 37°C and 5% CO2. After 24 h, the non-adherent cells were removed by changing the medium.Theculture medium was replaced three times per week. Whenthe cells were grown to confluence, the cells were lifted by0.25% trypsin and 0.02% EDTA in PBS. In this study, thecells were passed twice and were used for subsequentexperiments.

Prior to stereotactic transplantation, BMSC were incu-bated with 1-mg/mL bis-benzimide (Hoechst33342; Sigma,St Louis, MO, USA) over 24 h in order to fluorescent-label the nuclei. Subsequently, the Hoechst33342-labeledBMSC or vehicle was transplanted into the right striatumof SHR-SP at the age of 8 weeks, as described previ-ously.4,7 Briefly, the animals were anesthetized with 2.0%isoflurane in N2O : O2 (70 : 30). Under aseptic conditionson a clean bench, the animals were fixed to a stereotacticapparatus (Model DKI-900; David Kopf Instruments,Tujunga, CA, USA) and the cranium was exposedthrough midline skin incision. A burr hole was made3 mm right to the bregma, using a small dental drill. AHamilton syringe was inserted 5 mm into the brain paren-chyma from the surface of the dura mater, and 10 mL ofcell suspension (5 ¥ 105 cells) or 10 mL PBS was intro-duced into the right striatum over a period of 5 min, usingan automatic microinjection pump (Model KDS-310;Muromachi Kikai, Tokyo, Japan).

MRI

Using an MRI apparatus, the brain was visualized at theage of 11 weeks, as previously described.36 Thus, all MRIwere acquired using a small-animal, horizontal-bore, 7.0-Tesla MR scanner (Unity INOVA, Varian KInc., Cary,NC, USA) interfaced to a VNMR console (VNMR,Varian). The animals were anesthetized in the above-mentioned condition and were placed on non-magneticholder equipped with a nose cone for administration of

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anesthetic gas. A pilot scan of imaging sequence was usedfor reproducible positioning of the animal in the magnetat each MRI session. During the imaging procedure,anesthesia was maintained under the above-mentionedconditions, and core temperature was kept between 36.5and 37.0°C, using a feedback-controlled water bath. Todetect the lesion in the brain, T2- and T2-weighted imageswere obtained in all animals. Coronal T2-weighted imageswere acquired using standard two-dimensional Fourier-transform, multi-slice (9 slices, 1 mm thick) and spin echoT2 sequence. The sequence parameters were: repetitiontime (TR) = 2000 ms, echo time (TE) = 8 ms, numberof acquisitions (AC) = 8 times, field of view(FOV) = 50 ¥ 50 mm and matrix = 512 ¥ 256. The totalsequence time was approximately 68 min. CoronalT2-weighted images were acquired as two-dimensionalgradient echo imaging with TR of 500 ms, TE of 10 ms,flip angle of 15 degrees, number of AC of 4 times,50 ¥ 50 mm FOV, and 512 ¥ 256 image matrix. Slice thick-ness was 1 mm and slice separation was 0 mm. The totalsequence time was approximately 8 min.

Histological analysis

At 12 weeks of age, that is, 4 weeks after BMSC transplan-tation, the animals were sacrificed for histological analysis.In brief, they were deeply anesthetized with 4.0% isoflu-rane in N2O : O2 (70:30) and were transcardially perfusedwith 50 mL of heparinized saline, followed by 50 mL of 4%paraformaldehyde. The brain tissue was immersed in 4%paraformaldehyde for 2 days and embedded in paraffin.Then, 4-mm-thick coronal sections localized at the stereo-taxic level of the bregma were prepared for subsequentanalysis.

As the first step, the blue fluorescence signals that wereemitted from the nuclei of Hoechst33342-labeled cellswere identified using an appropriate filter on a fluores-cence microscope (BX51, Olympus, Tokyo, Japan) anda cooled charge-coupled device (CCD) camera (modelVB-6000/6010; Keyence Co., Osaka, Japan) equipped tothe microscope.

Next, the coronal sections were stained with HE. Toexamine the size of lateral ventricles, the sections werecaptured entirely at ¥40 magnification with a microscope,and then the images were digitally photographed usinga CCD camera equipped to the microscope (BZ-9000,Keyence Co., Japan). Subsequently, the areas of the lateralventricle and hemisphere were quantified using an imageanalyzing system (Image J version 1.43u, National insti-tutes of Health, USA). The area of lateral ventricle in eachhemisphere was presented as the percentage of lateral ven-tricle to the area of whole hemisphere. To quantitativelyanalyze the neuronal injury in the neocortex, 16 regions of

interest (ROIs, 350 mm ¥ 460 mm) were randomly assignedin the neocortex on both hemispheres. Photomicrographsof the entire ROI were obtained, and the intact and injuredneurons in each ROI were counted.The ratio of the injuredneurons to whole neurons was calculated in each animal.37

Finally, double fluorescence immunohistochemistrywas performed to visualize the parenchymal microvesselsand astrocytes. The deparaffinized sections were pro-cessed through antigen retrieval for 2 min by pressurepot. First, each coronal section was treated with rabbitanti-collagen IV antibody as the primary antibody (dilu-tion 1:500, no. 20441, Novotec, Saint Martin La Garenne,France) at 4°C overnight, and then was treated with anti-rabbit IgG-Rhodamine conjugated secondary antibody(dilution 1:200, AP132R, Millipore, Billerica, MA, US) atroom temperature for 60 min. Subsequently, the sectionswere treated with mouse antiGFAP antibody as theprimary antibody (dilution 1:500, No556327, BD Bio-science, Franklin Lakes, NJ, US) at room temperature for60 min, and anti-mouse IgG-Alexa Fluor488 conjugatedsecondary antibody (dilution 1:500, A11001, Invitrogen,Carlsbad, CA, US) was added at room temperature for60 min. The fluorescence emitted was observed throughan appropriate filter on a fluorescence microscope (BX51,Olympus, Tokyo, Japan) and digitally photographed usinga CCD camera (Model VB-6000/6010; Keyence Co.)equipped to the microscope. To analyze the density ofthe collagen IV-positive microvasculature, eight ROIs(680 mm ¥ 850 mm) in total were randomly assigned in theneocortex of both hemispheres. In addition, six ROIs(680 mm ¥ 850 mm) were also randomly assigned in thesubventricular zone (SVZ) on both hemispheres. Then,the area of collagen IV-positive blood vessels in eachROI was quantified, using the image analyzing system(Image J). Thus, the red fluorescence signal emitted fromthe microvasculature stained with anti-collagen IV anti-body was binarized (as black signal on white back-ground), quantified as the vascular fraction, and thenthe area of the collagen IV-positive blood vessels ineach ROI was presented as the percentage of collagenIV-positive area to the whole area of ROI. Finally, toquantitatively analyze the intact NVU in the neocortexand SVZ, the numbers of the normal microvasculaturesdoubly positive for collagen IV and GFAP were manuallycounted in each ROI.

Statistical analysis

All data were expressed as mean 1 SD. Continuous valueswere compared by unpaired t-test between two groups, andby one-factor analysis of variance (ANOVA) followed bypost hoc test among three groups. Values of P < 0.05 wereconsidered statistically significant.

BMSC transplantation & NVU protection 3


RESULTS

Physiological parameters in SHR-SP andWKY rats

All of WKY and SHR-SP were fed by stroke-permissivediet, and were loaded with 0.5% NaCl in drinking waterfrom the age of 9 weeks. All of them could survive withoutany behavioral evidence of stroke throughout the experi-ment. As shown in Table 1, both the BMSC- and vehicle-transplanted SHR-SP had significantly lower body weights(264 1 1.4 g and 270 1 15 g, respectively) than the WKY(373 1 9.3 g) at the age of 12 weeks (the BMSC-transplanted SHR-SP vs. the WKY, P < 0.01; the vehicle-transplanted SHR-SP vs. the WKY, P < 0.01). Bloodpressure of the BMSC- and vehicle-transplanted SHR-SPgradually elevated, while that of WKY did not changethroughout the experiment. Compared with the age-matched WKY, systolic and diastolic blood pressures weresignificantly higher in both the BMSC- and vehicle-transplanted SHR-SP at 6, 9 and 12 weeks of age (Table 1).

In vivo MRI study

After 3 weeks of stereotactic transplantation, in vivo MRIwas performed to monitor the morphological changes inthe cerebrum. As a result, both T2- and T2*-weightedimages (T2WI and T2*WI, respectively) did not demon-strate any evidence of cerebral infarct nor intracerebralhemorrhage in all animals. However, T2WI revealedabnormal dilatation of the lateral ventricles and peri-ventricular hyperintensity in the BMSC- or vehicle-transplanted SHR-SP, but not in the WKY. The ventriculardilatation was asymmetrical, but was observed on bothsides, suggesting that the stereotactic injection of BMSCor vehicle had no impact on ventricular dilatation(Fig. 1A–C).

Histological analysis

No neurological signs were observed in any animalthroughout the experiments. Grossly, their brains hadnormal morphology. On fluorescence microscopy, theHoechst33342-positive cells could easily be identified inthe right striatum of the BMSC-transplanted SHR-SP(Fig. 1D). However, the Hoechst33342-positive cellscould not be found in other brain regions, including theneocortex.

Coronal brain sections were stained with HE in allthree groups. The findings by lower magnifications werecomparable to those on MRI. Thus, neither cerebralinfarct nor intracerebral hemorrhage were observed inany animal group. However, as shown in Figure 2, amarked ventricle dilatation was observed in the SHR-SP.The area of the lateral ventricle in each hemisphere wasquantified. As a result, the vehicle-transplanted SHR-SPhad significantly larger lateral ventricle than the WKY,4.2 1 2.1% and 2.2 1 0.47%, respectively (P < 0.05).However, BMSC transplantation significantly preventedthe dilatation of the lateral ventricle in the SHR-SP.Thus, the BMSC-transplanted SHR-SP had significantlysmaller size of the lateral ventricle than the vehicle-transplanted SHR-SP, 2.2 1 1.3% and 4.2 1 2.1%, respec-tively (P < 0.05).

Although pathological changes in the small arteriolesare known to widely occur in SHR-SP more than 6 monthsof age,26,27,29 there was no evidence of such findings, includ-ing fibrinoid necrosis, wall thickening and narrowing/occlusion of the lumen in the neocortex and SVZ, in12-week-old SHR-SP (data not shown). On the other hand,selective neuronal damage was widely identified in theneocortex of the BMSC- or vehicle-transplanted SHR-SP,but not in the WKY as previously demonstrated.29 Thus, theshrunken neurons associated with dark cell change and

Table 1 Chronological changes of systolic blood pressure and body weight in Wistar-Kyoto rats (WKY) and salt-loaded stroke-pronespontaneously hypertensive rats (SHR-SP) with stereotactic transplantation of vehicle or bone marrow stromal cells (BMSC)

6 weeks 9 weeks 12 weeks

WKY SHR-SP SHR-SP WKY SHR-SP SHR-SP WKY SHR-SP SHR-SPvehicle BMSC vehicle BMSC Vehicle BMSC

Number of rats 3 4 3 3 4 3 3 4 3Systolic blood

pressure(mmHg)

113 1 9.3 142 1 9.0* 161 1 4.0** 116 1 3.8 177 1 6.8** 182 1 16** 120 1 5.1 228 1 15** 223 1 11**

Diastolic bloodpressure(mmHg)

80 1 2.0 106 1 24† 128 1 6.0†† 89 1 1.5 137 1 9.3†† 141 1 15†† 76 1 3 187 1 9.5†† 180 1 0.7††

Body weight(g)

160 1 6.4 141 1 7.9‡ 144 1 1.5 296 1 13 236 1 11‡‡ 229 1 5.7‡‡ 373 1 9.3 270 1 15‡‡ 264 1 1.4‡‡

* P < 0.05 (SHR-SP vehicle vs. WKY); ** P < 0.01 (SHR-SP vehicle or BMSC vs. WKY) in systolic pressure; † P < 0.05 (SHR-SP vehicle vs.WKY); †† P < 0.01 (SHR-SP vehicle or BMSC vs. WKY) in diastolic pressure; ‡ P < 0.05 (SHR-SP vehicle vs. WKY); ‡‡ P < 0.01 (SHR-SP vehicleor BMSC vs. WKY) in body weight.

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peri-neuronal vacuolation were observed in the neocortexof SHR-SP (Fig. 3A–C). The ratio of the damaged neuronsto the whole neurons in each ROI (350 mm ¥ 460 mm)was significantly higher in the vehicle- (0.27 1 0.13) andthe BMSC-transplanted SHR-SP (0.31 1 0.21) than inthe WKY (0.07 1 0.06; P < 0.001, respectively). Therefore,BMSC transplantation did not affect selective neuronaldamage in the SHR-SP (Fig. 3D).

Figures 4 and 5 show representative double fluores-cence immunohistochemistry against collagen IV andGFAP in the neocortex and SVZ, respectively. In thisstudy, the collagen IV-positive vessels with round, elliptical,

or linear morphology were defined as the parenchymalmicrovessels. Their diameter was 16 1 9.0 mm, 16 1 8.0 mmand 15 1 8.1 mm in the WKY, the vehicle-, and BMSC-transplanted SHR-SP, respectively. To clarify the underly-ing pathological process of abnormal ventricular dilatationand selective neuronal injury in the neocortex, their immu-noreactivity of basement membrane and their integritybetween the basement membrane and astrocyte end-feetin the parenchymal microvessels were examined. As aresult, the density of the collagen IV-positive microvesselsin each ROI of the neocortex was significantly lower in thevehicle-transplanted SHR-SP (1.3 1 0.92%) than in the

Fig. 1 Representative coronal spin echoT2-weighted images of Wistar-Kyoto rats(WKY) (A), the vehicle-transplanted stroke-prone spontaneously hypertensive rats(SHR-SP) (B) and the Hoechst33342-labeledbone marrow stromal cells (BMSC)-transplanted SHR-SP (C) at the age of 11weeks. Note, WKY had no evidence of eithercerebral infarct or intracerebral hemorrhage(A). In vehicle- and BMSC-transplantedSHR-SP, asymmetrical dilatation of thelateral ventricle was demonstrated (B and C,respectively). Boxed area showing fluores-cence microphotography for distributionof the transplanted BMSC labeled byHoechst33342 (D). Photomicrographs showthe engraftment of Hoechst33342-positivecells in BMSC-transplanted SHR-SP (arrowsin panel D). Scale bars = 2 mm.

Fig. 2 Photomicrographs of HE stainshowing coronal brain sections of all threegroups at the stereotaxic level of the bregma.Consistent with MRI findings, neither cere-bral infarcts nor intracerebral hemorrhagesare evident in all groups. However, a markedventricle dilatation was observed in thevehicle-transplanted stroke-prone spontane-ously hypertensive rats (SHR-SP) (B) com-pared with Wistar-Kyoto rats (WKY) (A). Incontrast, the bone marrow stromal cells(BMSC)-transplanted SHR-SP had signifi-cantly smaller size of the lateral ventricle(C) than the vehicle-transplanted SHR-SP.Scatter plotting shows the fraction of lateralventricle in each hemisphere of the threeanimal groups (D). Significant difference isdetected in the vehicle-transplanted SHR-SP(*P < 0.05, one-factor analysis of variancefollowed by Fisher’s protest least significantdifference test among three groups). Scalebar = 5 mm.



WKY rats (3.9 1 0.87%, P < 0.01). However, BMSC trans-plantation significantly attenuated the decrease in the col-lagen IV-positive microvessels in the neocortex. Thus, thedensity of the collagen IV-positive microvessels in theBMSC-transplanted SHR-SP (3.5 1 1.4%) was signifi-cantly higher than in the vehicle-transplanted SHR-SP(1.3 1 0.92%, P < 0.01), and was comparable to that of theWKY (3.9 1 0.87%, Fig. 6A). The findings in the SVZ weresimilar to those in the neocortex (Fig. 6B). In addition,there was no apparent astrocytic proliferation associatedwith selective neuronal damage in the neocortex ofSHR-SP.

The merged images of double fluorescence immunohis-tochemistry against collagen IV and GFAP demonstratedthat the integrity between basement membrane and astro-cyte end-feet were kept intact in the neocortex of the WKY(Fig. 4A–D).The number of these normal microvessels wascounted in each ROI randomly assigned in the neocortex.As a result, the number of normal microvessels in theneocortex was significantly smaller in the vehicle-transplanted SHR-SP than in the WKY, 11 1 7.3/mm2

and 26 1 6.8/mm2, respectively (P < 0.01). BMSC trans-plantation significantly prevented decrease in normalmicrovessels in the neocortex. Thus, the number ofnormal microvessels in the neocortex was significantlylarger in the BMSC-transplanted SHR-SP than inthe vehicle-transplanted SHR-SP, 23 1 14/mm2 and 11 17.3/mm2, respectively (P < 0.01, Fig. 6C). The findingsin the SVZ were similar to those in the neocortex(Fig. 6D).

DISCUSSION

Vascular and neuronal damage in SHR-SP

The principal findings reported here are, first, that thelateral ventricles are dilated and the neocortical neuronsare selectively damaged in 12-week-old SHR-SP. The find-ings are observed prior to the development of cerebralinfarct and intracerebral hemorrhage. Simultaneously, thenumber of collagen IV-positive microvessels starts todecrease, and the integrity of NVUs are disrupted in theneocortex and SVZ.

Previous works have revealed that blood pressure spon-taneously starts to increase at 4 weeks of age in maleSHR-SP and reach higher than 220 mmHg at 20 weeks ofage. The incidence of ischemic or hemorrhagic lesions israre at younger than 12 weeks of age, but exceeds 80% inmales over 100 days of age.24 However, the loading of high-salt diet is known to accelerate these pathological changes.They rapidly develop the lesions when fed by 1–4% NaClat 4–6 weeks of age. Thus, Smeda et al. demonstrated anacceleration of stroke development (mean age at death15.3 1 0.5 weeks) in SHR-SP fed a diet containing 4%NaCl.38 Blezer et al. observed cerebral edema with T2WIin 1% NaCl-loaded SHR-SP and monitored them untilnatural death. As a result, median survival was 54 daysafter start of salt loading and the terminal level of cerebraledema was 19.0 1 3.0%.25 Sironi et al. monitored brainlesions in salt-loaded SHR-SP, using MRI and histologicalanalysis. They found that salt-loaded SHR-SP developed

Fig. 3 Photomicrographs of the neocortexof the same HE stain in three animal groupsdemonstrate selectively injured neurons(arrows). Scatter plotting shows the ratio ofinjured neurons to whole neurons in eachregion of interest (ROI) (D). Significantdifferences are detected in both groupsof stroke-prone spontaneously hypertensiverats (SHR-SP) compared with Wistar-Kyotorats (WKY) (**P < 0.001, one-factor analysisof variance followed by Bonferroni’s testamong three groups). Scale bar = 125 mm.

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cerebral legions at a median of 42 1 3 days from the start ofsalt loading and their brains were characterized by tissuethinning due to loss of neuronal cells.39

In this study, therefore, we started to load 0.5% NaCl-containing water from the age of 9 weeks to mildly accel-erate their brain lesions. As a result, MRI and histologicalanalysis revealed neither cerebral infarct nor intracerebralhemorrhage at 12 weeks of age. Instead, the ventriculardilatation and selective neuronal damage were observed intheir brains. Previously, only Tajima et al. evaluated thebrain structure of 6- to 7-month-old SHR and reported thatventricular volume was twofold greater in SHR than inWKY rats.40 Therefore, this is the first presentation thatdenotes these degenerative processes in SHR-SP. The

pathological mechanisms underlying these processes arestill obscure. Therefore, we examined the changes ofthe parenchymal microvessels in the brains of SHR-SP,because the main target of hypertension is known as paren-chymal microvessels. Then, double fluorescence immuno-histochemistry clearly demonstrates that the number of thecollagen IV-positive microvessels significantly decreasesand the integrity of the NVU is disrupted in the neocortexand SVZ at 12 weeks of age. Type-IV collagen is one of themajor functional components of the basement membranesthat are intercalated between the endothelium and astro-cyte end-feet.41 The basement membranes of cerebralmicrovessels are known to provide a scaffold on which theendothelial and glial compartments interact with each

A

E

I J K L

F G H

B C D

Fig. 4 Photomicrographs of double fluorescence immunohistochemistry against collagen IV (A, E, I) and GFAP (B, F, J) in the neocortexof Wistar-Kyoto rats (WKY) (A–D), vehicle- (E–H) and the bone marrow stromal cells (BMSC)-transplanted stroke-prone spontaneouslyhypertensive rats (SHR-SP) (I–L) at the age of 12 weeks. Photomicrographs shown in panels A, E and I illustrate the density of collagenIV-positive microvasculature in all three groups. Low- (C, G, K) and high-power magnification photomicrographs (D, H, L) of mergedimages show the distribution of normal microvasculature (arrows in panel D, H, L) that keeps its integrity between basement membraneand astrocyte end-feet in the three groups. Scale bars = 100 mm.



other.42 A previous study has demonstrated the increaseddensity of type-IV collagen-positive capillaries in thebrains of young SHR-SP, suggesting an association withtheir hypertrophy.43 This discrepancy may result fromdifferences in experimental protocol. Thus, Ritz et al.observed the brains of 2- to 9-month-old SHR-SP withoutsalt loading and quantified collagen IV-positive capillarydensity in their cortex and putamen.As a result, they foundthat collagen IV-positive capillary density was significantlyincreased in the neocortex of only 2-month-old SHR-SP,when compared with normotensive WKY. In contrast, thisstudy employs salt-loaded SHR-SP and demonstrates thedecreased density of collagen IV-positive microvessels inthe brains of 12-week-old SHR-SP. Therefore, several con-

ditions such as age and salt loading may largely influencepathological changes in their brain microvessels. SHR-SPwithout NaCl treatment would be desirable to include asthe control to assess the influence of salt loading againstthese pathological processes as the next step.

More interestingly, this study clearly shows that theintegrity of NVU is simultaneously disrupted in the brainsof 12-week-old SHR-SP prior to the development of cere-bral infarct or intracerebral hemorrhage. This fact may beconsistent with the previous findings that cerebral bloodflow (CBF) autoregulation is impaired prior to the devel-opment of cerebral stroke in SHR-SP fed a high-salt diet.Thus, Smeda et al. measured CBF in 13-week-old, stroke-free SHR-SP that were fed by a diet containing 4% NaCl,

A B C D

E F G H

I J K L

Fig. 5 Photomicrographs of double fluorescence immunohistochemistry against collagen IV (A, E, I) and GFAP (B, F, J) in thesubventricular zone and adjacent striatum of Wistar-Kyoto rats (WKY) (A–D), vehicle- (E–H) and the bone marrow stromal cells(BMSC)-transplanted stroke-prone spontaneously hypertensive rats (SHR-SP) (I-L) at the age of 12 weeks. Photomicrographs shown inpanels A, E and I illustrate the density of collagen IV-positive microvasculature in all three groups. The merged images (D, H, L) show thedistribution of normal microvasculature (arrows in panel D, H, L) that keeps its integrity between basement membrane and astrocyteend-feet in three groups. Scale bars = 125 mm.

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and found the loss of CBF autoregulation prior to strokedevelopment.44 Their findings may correlate well with ourpresent results that the integrity of the NVU is disturbedprior to the development of cerebral stroke. Nowadays, invitro studies have indicated the importance of microvessel–neuron interactions in physiological and pathological con-ditions. Thus, del Zoppo has proposed the concept thatNVU consists of microvessels (endothelial cells, basallamina matrix, astrocyte end-feet and pericytes), astro-cytes, neurons and their axons, and other supporting cellssuch as microglia and oligodendroglia, and that all of themmodulate the function of the “unit”. This concept providesa framework for considering bi-directional communicationbetween neurons and their supply microvessels with theparticipation of the intervening astrocytes. NVU may alsooffer a platform for understanding the evolution of CNSinjury processes. Moreover, its entire protection mayprovide effective therapies for ischemic stroke.34,42 Basedon these observations, the present results strongly suggestthat long-lasting hypertension may damage the integrity ofthe NVU and then selectively neocortical neurons, leadingto ventricular dilatation prior to the occurrence of cerebralinfarct or intracerebral hemorrhage.

Effects of BMSC transplantation on degenerativechanges in SHR-SP

Another exciting finding of this study is the fact thatengrafted BMSC significantly ameliorate these pathologi-cal processes in SHR-SP, including the breakdown of

microvascular integrity and ventricular dilatation, whendirectly transplanted into their brains. As aforementioned,a growing number of studies have demonstrated thatBMSC protect and repair damaged CNS tissue throughmultiple mechanisms. There are several explanations forthis, although it is not fully understood. Of these, BMSCper se are believed to differentiate into neural cells in thehost’s brain (“transdifferentiation theory”).8–14 The BMSCalso produce some neuroprotective or neurotrophic factorsand support the survival of the host neural cells (“thefeeder theory”).20–23 Moreover, previous studies demon-strated that the transplanted BMSC promote VEGF secre-tion, VEGF receptor-2 expression and angiogenesis in theischemic brain after stroke.18,19 In this study, BMSC werelabeled with Hoechst33342 and were injected into the rightstriatum. Hoechst-positive cells could be found in the ipsi-lateral striatum, that is, the injection site at 4 weeks aftertransplantation, but not in other regions. The BMSC-transplanted SHR-SP had significantly larger numbers ofnormal microvessels with intact integrity than the vehicle-transplanted SHR-SP. Therefore, it is unlikely that thetransplanted cells may protect the microvessels in SHR-SPby differentiating into neural cells or vascular cells. Thepresent results strongly suggest that BMSC may surviveand protect the integrity of NVU through releasing someangiogenic or vascular protective factors or by upregulat-ing the production of endogenous VEGF and VEGFreceptors indirectly,18 when directly transplanted into thebrain of 8-week-old SHR-SP. Moreover, their protectiveeffects on NVU may further ameliorate hypertension-

Fig. 6 Scatter plot graphs showing thequantification of the collagen IV signal (A,B) and the number of collagen IV–GFAPdouble positive microvessels (C, D) inWistar-Kyoto rats (WKY), vehicle- and thebone marrow stromal cells (BMSC)-transplanted stroke-prone spontaneouslyhypertensive rats (SHR-SP) are presented.Graph (A) and (C) demonstrate these valuesin 16 regions of interest (ROIs) of the neo-cortex in both hemispheres (circles in theupper boxed area). Significant differencesare detected in WKY and the BMSC-transplanted SHR-SP compared withvehicle-transplanted SHR-SP (**P < 0.001,one-factor analysis of variance [ANOVA]followed by Bonferroni’s test among threegroups). Similarly, graphs (B) and (D) dem-onstrate these values in 12 ROIs of thesubventricular zone (SVZ) and adjacentstriatum (circles in the lower boxed area) andsignificant differences are also detected inWKY and the BMSC-transplanted SHR-SPcompared with vehicle-transplanted SHR-SP(**P < 0.001, one-factor ANOVA followedby Bonferroni’s test among three groups).



related brain atrophy and ventricular dilatation, althoughBMSC cannot prevent selective neuronal damage in theneocortex. However, it is still obscure whether BMSCtransplantation could inhibit or delay the occurrence ofcerebrovascular events and improve neurologic functionsin SHR-SP, because they were sacrificed prior to the onsetof any symptoms in this preliminary study. A possible neu-rologic improvement in the transplanted animals should beundertaken in the next study.

For decades, BMSC have been studied in order toregenerate damaged CNS tissue and to enhance functionalrecovery after various kinds of CNS disorders, includingischemic stroke.2 Preliminary clinical trials have alreadybeen started to test the safety and feasibility of BMSCtransplantation.45,46 However, this study strongly indicatesanother attractive aspect of BMSC that may also be usefulto protect brain microvessels and parenchyma in hyperten-sive patients at higher risk for cerebral stroke. Thus, theBMSC may be a practical cell source for prophylactic aswell as restorative medicine. However, it should be remem-bered that the underlying mechanisms of their prophylac-tic actions are still obscure and further studies would bewarranted to reach final conclusions. In addition, BMSCwere stereotactically transplanted into the brain in thisstudy. However, less invasive delivery would be warranted,when considering the clinical application of BMSC trans-plantation as prophylactic therapy.

In conclusion, the present results strongly suggest thatlong-lasting hypertension may primarily damage NVU andneurons, leading to tissue atrophy and ventricular dilata-tion prior to the occurrence of cerebral stroke. Thephenomenon would provide important information onetiology of hypertension-induced degenerative changes inthe brain. Furthermore, BMSC may ameliorate these dam-aging processes when directly transplanted into the brain,opening the possibility of prophylactic medicine to preventmicrovascular and parenchymal damaging processes inhypertensive patients.

ACKNOWLEDGEMENTS

This study was supported by Grant-in-aid from theMinistry of Education, Science and Culture ofJapan (No.20591701, No.20390377, No.21390400, andNo.23390342). The authors sincerely thank YumikoShinohe for her technical assistance.

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Transplanted bone marrow stromal cells protect neurovascular units and ameliorate brain damage in stroke-prone spontaneously hypertensive rats