Intravenous infusion of mesenchymal stem cells inhibits … · epeRfusion therapy with intravenous recombinant tissue plasminogen activator (rtPA) administra-tion is the international

LABORATORY INVESTIGATIONJ Neurosurg 127:917–926, 2017

RepeRfusion therapy with intravenous recombinant tissue plasminogen activator (rtPA) administra-tion is the international standard for acute ischemic

stroke; however, hemorrhagic complications after rtPA therapy are a major problem that may result in unfavorable

clinical outcomes.35 Only a small percentage of stroke pa-tients can receive rtPA therapy because of the narrow ther-apeutic time window due to elevated risks of symptomatic intracranial hemorrhage. Thus, to suppress the incidence of hemorrhagic events following intravenous rtPA therapy

ABBREVIATIONS BBB = blood-brain barrier; DWI = diffusion-weighted imaging; EvB = Evans Blue; GFP = green fluorescent protein; IL = interleukin; MCA = middle cere-bral artery; MMP = matrix metalloproteinase; MSC = mesenchymal stem cell; NF-kb = nuclear factor kappa light-chain enhancer of activated B cells; NS = normal saline; PBS = phosphate-buffered saline; PWI = perfusion-weighted imaging; rCBF = regional cerebral blood flow; ROI = region of interest; rtPA = recombinant tissue plasminogen activator; tMCAO = transient MCA occlusion; TNF = tumor necrosis factor. SUBMITTED February 1, 2016. ACCEPTED August 24, 2016.INCLUDE WHEN CITING Published online January 6, 2017; DOI: 10.3171/2016.8.JNS16240.

Intravenous infusion of mesenchymal stem cells inhibits intracranial hemorrhage after recombinant tissue plasminogen activator therapy for transient middle cerebral artery occlusion in ratsMasahito Nakazaki, MD,1 Masanori Sasaki, MD, PhD,1,3,4 Yuko Kataoka-Sasaki, MD, PhD,1 Shinichi Oka, MD, PhD,1 Takahiro Namioka, MD,1 Ai Namioka, MD,1 Rie Onodera, PhD,1 Junpei Suzuki, PhD,1 Yuichi Sasaki, PhD,1 Hiroshi Nagahama, BS,1 Takeshi Mikami, MD, PhD,2 Masahiko Wanibuchi, MD, PhD,2 Jeffery D. Kocsis, PhD,3,4 and Osamu Honmou, MD, PhD1,3,4

1Department of Neural Regenerative Medicine, Research Institute for Frontier Medicine, and 2Department of Neurosurgery, Sapporo Medical University School of Medicine, Sapporo, Japan; 3Department of Neurology, Yale University School of Medicine, New Haven, Connecticut; and 4Center for Neuroscience and Regeneration Research, VA Connecticut Healthcare System, West Haven, Connecticut

OBJECTIVE Reperfusion therapy with intravenous recombinant tissue plasminogen activator (rtPA) is the standard of care for acute ischemic stroke. However, hemorrhagic complications can result. Intravenous infusion of mesenchymal stem cells (MSCs) reduces stroke volume and improves behavioral function in experimental stroke models. One sug-gested therapeutic mechanism is inhibition of vascular endothelial dysfunction. The objective of this study was to de-termine whether MSCs suppress hemorrhagic events after rtPA therapy in the acute phase of transient middle cerebral artery occlusion (tMCAO) in rats.METHODS After induction of tMCAO, 4 groups were studied: 1) normal saline [NS]+vehicle, 2) rtPA+vehicle, 3) NS+MSCs, and 4) rtPA+MSCs. The incidence rate of intracerebral hemorrhage, both hemorrhagic and ischemic volume, and behavioral performance were examined. Matrix metalloproteinase–9 (MMP-9) levels in the brain were assessed with zymography. Quantitative analysis of regional cerebral blood flow (rCBF) was performed to assess hemodynamic change in the ischemic lesion. RESULTS The MSC-treated groups (Groups 3 and 4) experienced a greater reduction in the incidence rate of intrace-rebral hemorrhage and hemorrhagic volume 1 day after tMCAO even if rtPA was received. The application of rtPA en-hanced activation of MMP-9, but MSCs inhibited MMP-9 activation. Behavioral testing indicated that both MSC-infused groups had greater improvement than non-MSC groups had, but rtPA+MSCs provided greater improvement than MSCs alone. The rCBF ratio of rtPA groups (Groups 2 and 4) was similar at 2 hours after reperfusion of tMCAO, but both were greater than that in non-rtPA groups.CONCLUSIONS Infused MSCs may inhibit endothelial dysfunction to suppress hemorrhagic events and facilitate func-tional outcome. Combined therapy of infused MSCs after rtPA therapy facilitated early behavioral recovery.https://thejns.org/doi/abs/10.3171/2016.8.JNS16240KEY WORDS transplantation; mesenchymal stem cell; stroke; intracranial hemorrhage; recombinant tissue plasminogen activator; rodent; middle cerebral artery occlusion

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and to extend the therapeutic time window, development of an adjunctive therapy to inhibit endothelial dysfunction would be desirable.

Systemic administration of mesenchymal stem cells (MSCs) after cerebral infarction can reduce stroke vol-ume and improve behavioral function in experimental stroke models.3,6,9,15,19 Suggested therapeutic mechanisms of MSCs in various models of CNS diseases include secre-tion of neurotrophic factors that provide neuroprotection, neurogenesis,8 induction of axonal sprouting, neovascular-ization, and immunomodulation.28,31 In addition to these mechanisms, recent studies have reported that MSCs have the potential to maintain blood-brain barrier (BBB) in-tegrity and reduce BBB leakage that could contribute to their therapeutic efficacy.17 Several clinical studies using intravenous infusion of MSCs in stroke patients have been performed or are ongoing.7,8

In the present study, we tested the hypothesis that in-travenous infusion of MSCs inhibits vascular endothelial dysfunction decreasing both the incidence rate of intra-cerebral hemorrhage and hemorrhagic volume after rtPA therapy using a transient (90-minute) middle cerebral ar-tery occlusion (tMCAO) model in rats. Quantitative analy-sis of regional cerebral blood flow (rCBF) was performed to assess hemodynamic changes in the ischemic lesion. Additionally, ischemic volume and behavioral perfor-mance were recorded during the study period.

MethodsAnimals

The use of animals in this study was approved by the animal care and use committee of Sapporo Medical Uni-versity, and all procedures were carried out in accordance with institutional guidelines. All MRI evaluations, zymog-raphy, behavioral testing, and statistical analyses were per-formed by persons who were naïve with respect to treat-ment condition.

Preparation of MSCs From Rat Bone MarrowThe methodology of MSC culture was based on our

previous studies.11,32 Briefly, bone marrow was obtained from femoral bones in adult Sprague-Dawley rats or green fluorescent protein (GFP)-expressing Sprague-Dawley rats (W-Tg [CAG-GFP]184Ys) diluted to 15 ml with DMEM (Sigma) supplemented with 10% heat-inactivated fetal bo-vine serum; Thermo Fisher Scientific Inc.), 2 mM l-glu-tamine (Sigma), 100 U/ml penicillin, 0.1 mg/ml strepto-mycin (Thermo Fisher Scientific Inc.) and incubated for 3 days (5% CO2, 37°C). When cultures almost reached confluence, the adherent cells were detached with trypsin–ethylenediaminetetraacetic acid solution (Sigma) and sub-cultured at 1 × 104 cells/ml. In the present study, we used MSCs after 3 passages.

Cerebral Ischemic ModelWe induced tMCAO for 90 minutes by using a previ-

ously described method of intraluminal vascular occlu-sion.6,9,27 Adult male Sprague-Dawley rats weighing 280–330 g (n = 222) were anesthetized with an intraperitoneal injection of ketamine (75 mg/kg) and xylazine (10 mg/kg).

A 20.0- to 22.0-mm length of 3–0 surgical Monosof su-ture (Covidien), with the tip rounded by heating it near a flame, was advanced from the external carotid artery into the lumen of the internal carotid artery until it blocked the origin of the MCA for 90 minutes with initial diffusion-weighted imaging (DWI)-MRI performed at the 60-min-ute occlusion period. The initial stroke volume evaluated with DWI was standardized at 220–370 mm3 and excluded animals outside this range to assess the efficacy of MSCs following rtPA for a large ischemic injury.30 We used a suture occlusion/reperfusion model in this study to inves-tigate the effects of rtPA and MSCs following complete reperfusion. Physiological parameters (rectal temperature, blood pH, partial pressure of oxygen, partial pressure of carbon dioxide, and blood pressure) were maintained within normal ranges during surgery and transplantation procedures for all animals and did not significantly differ among the experimental groups.9

Experimental ProtocolsThe experimental protocol is shown in Fig. 1. Sixty

minutes after establishing tMCAO, DWI was obtained to evaluate initial stroke volume. Thirty minutes after DWI, the inserted surgical Monosof suture was removed for reperfusion. Then, the rats were randomized into 4 ex-perimental groups. In Group 1 (1 ml normal saline [NS] + 1 ml vehicle [DMEM]; n = 55), rats were injected in-travenously with 1 ml NS immediately after reperfusion, then injected with vehicle after 30 minutes. In Group 2 (rtPA+vehicle; n = 68), rats were injected intravenously with rtPA (10 mg/kg; 1 ml) immediately after reperfusion, then injected with vehicle after 30 minutes. In Group 3 (NS+MSCs; n = 51), rats were injected intravenously with NS (1 ml) immediately after reperfusion, then were in-jected with MSCs (1.0 × 106 cells each) in 1 ml DMEM after 30 minutes. In Group 4 (rtPA+MSCs; n = 48), rats were injected intravenously with rtPA (10 mg/kg; 1 ml) immediately after reperfusion, then injected with MSCs (1.0 × 106 cells each) in 1 ml DMEM after 30 minutes. All rats were injected daily with cyclosporine A (10 mg/kg, in-traperitoneally).1,5,16,20,22,28,37 All intravenous infusions were delivered through the left femoral vein.

MRI Studies and Measurement of Infarct VolumeRats were anesthetized with ketamine (75 mg/kg, in-

traperitoneally) and xylazine (10 mg/kg, intraperitone-ally). Each rat was placed in an animal holder/MRI probe apparatus and positioned inside the magnet. The animal’s head was held in place inside the imaging coil. All MRI measurements were performed using a 7-T, 18-cm bore superconducting magnet (Oxford Magnet Technologies) interfaced to a UNITY INOVA console (Oxford Instru-ments and Varian Inc.) as described previously.6,12

Briefly, DW images were obtained from a 1.0-mm-thick coronal section with a 0.5-mm gap using a 30 mm × 30 mm field of view (TR 3000 msec, TE 37 msec, b value 1000 sec/mm2) and reconstructed using a 128 × 128 im-age matrix. The T2-weighted images were obtained from a 1.0-mm-thick coronal section with a 0.5-mm gap using a 30 mm × 30 mm field of view (TR 3000 msec, TE 30


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msec) and reconstructed using a 256 × 256 image matrix. Accurate positioning of the brain was performed to center the image slice 5 mm posterior to the rhinal fissure with the head of the rat held in a flat skull position.

DW images were obtained 60 minutes after tMCAO, and T2-weighted images were obtained at 1, 4, 7, 14, 21, and 28 days after tMCAO. The ischemic lesion area was calculated from MR images using imaging software (Sci-on Image, beta version 4.0.2, Scion Corporation) based on the previously described method.18 Lesion volume (mm3) was determined by analysis of high-intensity areas on 9 serial coronal images collected through the cerebrum. For each slice, the higher-intensity lesions on DWI and T2-weighted imaging, where the signal intensity was 1.25 times higher than the counterpart in the contralat-eral brain lesion, were marked as the ischemic lesion area, and infarct volume was calculated taking slice thickness (1 mm/slice) into account. The presence of intracerebral hemorrhage was counted when there was a low-intensity area in the T2-weighted imaging section.

Perfusion-weighted imaging (PWI) was acquired us-ing a T2*-weighted (TR = 13 msec, TE = 6.0 msec) gradi-ent echo sequence.20,32 Four rats in each group were used. A dynamic image series of 30 measurements resulted in a total scan time of 26 seconds, with a field of view of 30 mm and image acquisition matrix of 128 × 64, which was interpolated by zero-filling to 512 × 512. During the dynamic series, a triple-dose (0.6 ml/kg) bolus injection of Magnevist (Schering AG) was started after the fifth acquired volume to ensure a sufficient precontrast base-line. Images were reconstructed using Innova Vision (GE Healthcare). PWI measurements were obtained 2 hours after reperfusion of tMCAO. For the PWI-derived CBF maps, 1 coronal slice at bregma -0.4 mm34 was chosen for cerebral blood flow quantification as described previ-ously.20,32 The regions of interests (ROIs, 60 × 60 pixels) were placed in the ischemic hemisphere. The coordinate relative to the bregma for the center of ROI was on the DWI: 0.3 mm caudal, 1.25 mm lateral, 0.1 mm depth.32 The rCBF in each ROI was quantified using Perfusion Solver software.20,32 An rCBF ratio was calculated with the rCBF in the infarcted hemisphere divided by that of the noninfarcted hemisphere.

Histological Processing for GFPFor immunohistological analysis, 4 rats in each group

were used. Rats were deeply anesthetized with ketamine and xylazine (75/10 mg/kg, intraperitoneally), perfused with saline and 0.1 M phosphate buffer, and processed for standard frozen sectioning. Cryosections were cut us-ing a cryostat and mounted on glass slides. Sections were washed in phosphate-buffered saline (PBS) and 0.1% Tween 20 three times, blocked in 5% normal donkey se-rum and 0.3% Triton X-100 in PBS at room temperature for 30 minutes, and then incubated overnight in primary antibodies diluted with 5% normal donkey serum, 0.3% Triton X-100, and PBS at 4°C. The cryosections were processed for immunolabeling for chicken polyclonal anti-antibody (20 mm thick, 1:1000; Abcam, ab13970) to detect GFP. After being washed in PBS-Tween 20 four times, sections were incubated in secondary antibody AF 488-conjugated goat anti–chicken immunoglobulin Y for GFP (1:1000; Abcam, 150173), counterstained with DAPI, and coverslipped with VECTASHIELD (Vector Labora-tories). The frozen sections were examined using confocal microscopy with Ex/Em (405 nm/410–502 nm for DAPI; 488 nm/499–553 nm for GFP; LSM780 ELYRA S.1 sys-tem, Zeiss).

Gelatin ZymographyGelatin zymography was performed using brain tis-

sue removed from rats on Day 1 after tMCAO, as pre-viously described.13 Four rats in each group were used. Briefly, brain samples were homogenized immediately after perfusion in ×10 volume lysis buffer (150 mM NaCl, 1% sodium dodecyl sulfate, 0.1% deoxycholic acid, and 50 mM Tris-HCl, pH 7.5) containing protease inhibitors on ice. After centrifugation at 9000g for 15 minutes at 4°C, the supernatant was collected and stored at -80°C until use. Total protein concentration of each supernatant was determined by Thermo BCA protein assay (Pierce). The activity of matrix metalloproteinase–9 (MMP-9) in each sample was measured using a gelatin-zymography kit (Primary Cell), according to the manufacturer’s in-structions. In brief, each sample containing 20 mg of pro-tein was diluted with the homogenizing buffer in the kit,

FIG. 1. Experimental protocol. DWIs were obtained 60 minutes after tMCAO. The tMCAO rats were randomized to 4 groups and reperfused, then infused (with normal saline or rtPA) after 90 minutes of tMCAO induction. After 30 minutes of reperfusion, they received medium (vehicle) or MSCs intravenously.


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mixed with an equal volume of sample buffer, and loaded for electrophoresis for 2 hours. The gels were washed and incubated for 48 hours in incubation buffer at 37°C, then stained with Coomassie blue and scanned. To measure band intensities, densitometric analysis was performed using TotalLab Quant.

Evans Blue AnalysisFour rats in each group were used for Evans Blue

(EvB) analysis. Rats were anesthetized with ketamine and xylazine (75/10 mg/kg, intraperitoneally). The 4% EvB, which was dissolved in 0.9% saline, was injected as a sin-gle bolus dose of 1 ml/kg via the right femoral vein after infusion of MSCs or vehicle. At Day 1, rats were deeply anesthetized with ketamine and xylazine (75/10 mg/kg, intraperitoneally) and transcardially perfused with ice-cold PBS to remove the intravascular dye. The whole brains were removed, and the macroscopic images were acquired using a Nikon AZ100 microscope with a ×0.5 objective. The whole brains were dissected into coronal 1-mm sections using a vibratome. The coronal slices were examined with a Nikon AZ100 microscope with a ×0.5 objective.

For extraction to quantify the total EvB content in the tissue, after taking the macroscopic images, the brains were placed in 1:2 weight (mg):volume (ml) ratios of 0.9% saline and homogenized for 1 minute using a sonifier (Branson Ultrasonic Corporation). Samples were incu-bated in 37°C for 60 minutes, then ×2 weight of 50% was added in each sample and homogenized for 1 minute with a beam homogenizer. Homogenized samples were centri-fuged at 10,000g for 20 minutes to remove tissue debris, and the supernatants were added to a 96-well plate (30 ml per well, each plate supplemented with 90 ml of 95% etha-nol and thoroughly mixed) for fluorescence spectroscopy (620 nm/680 nm).

Treadmill Stress TestRats were trained 20 min/day for 2 days a week to run

on a motor-driven treadmill (Muromachi Inc.) at a speed of 20 m/min with a slope of 20° before tMCAO. The max-imum speed at which the rats could run on a motor-driven treadmill was recorded on Days 1, 4, 7, 14, 21, and 28 after tMCAO.9,20,30,36

Statistical AnalysisAll statistical analyses were performed using JMP 11.1

for Windows (SAS Institute Inc.). Categorical data in con-tingency tables were analyzed using the chi-square test and, if appropriate, the Bonferroni test for post hoc analy-sis to determine any significant differences between each pair of groups. Continuous data were assessed for nor-mality using the Shapiro-Wilk test; normally distributed continuous data were analyzed by 1-way ANOVA, and the Tukey-Kramer test was used to compare the subgroups if significance was found. Continuous data that did not pass this normality test were compared using the Kruskal-Wal-lis test, and the Steel-Dwass test was used to compare the subgroups if significance was found.

ResultsHemorrhagic Events After Intravenous rtPA

Representative images are shown in Fig. 2A for NS+vehicle (Group 1), rtPA+vehicle (Group 2), NS+MSCs (Group 3), and rtPA+MSCs (Group 4). Low-intensity areas suggesting hemorrhage were detected in the T2-weighted imaging in all groups. Quantitative analysis demonstrated that acute hemorrhagic events in Group 2 were higher than those in the other 3 experimental groups in terms of incidence rate (Group 1: 9.5%; Group 2: 50%; Group 3: 3.6%; Group 4: 10%, p < 0.01, chi-square test; Fig. 2B) and hemorrhagic volume (Group 1: 0.14 ± 0.01 mm3; Group 2: 3.20 ± 1.54 mm3; Group 3: 0.01 ± 0.01 mm3; Group 4: 0.48 ± 0.39 mm3, p < 0.01, Kruskal-Wallis test; Fig. 2C). It should be noted that hemorrhagic events after rtPA thera-py in Group 2 were reduced by MSC infusion in Group 4 (40% reduction, p < 0.01, chi-square test; Bonferroni test for post hoc: volume 2.72 mm3 reduction, p < 0.05, Steel-Dwass test) suggesting that intravenous infusion of MSCs has the potential to reduce the incidence and volume of intracerebral hemorrhage induced by rtPA therapy.

Detection of GFP-MSCs In VivoHistological analysis at 1 day after GFP-MSC (green)

infusion indicated that the infused cells survived and were distributed to the infarcted hemisphere (Fig. 2D). There were few GFP-MSCs in the contralateral hemisphere. To determine whether autofluorescence of the MSCs was present at the wavelengths used to study GFP fluorescence, we examined these sections of animals infused with MSCs derived from wild-type Sprague-Dawley rats. No green cells were observed in these lesions (data not shown).

Gelatin ZymographyMMP-9 contributes to hemorrhagic transformation af-

ter reperfusion.10 Gelatin zymography indicated that on the ipsilateral (lesion) side of the brain, MMP-9 activity was high in Groups 1 and 2 (NS+vehicle and rtPA+vehicle, respectively). However, when MSCs were infused with (Group 4) and without rtPA (Group 3), MMP-9 activity was low (Fig. 3). It should be noted that the rtPA+MSCs group (Group 4) had significantly lower MMP-9 activity than the rtPA+vehicle group (Group 2; p < 0.01, Tukey’s post hoc test) had, and MMP-9 activity in the NS+MSCs group (Group 3) was significantly lower than the NS+vehicle group (Group 1 [NS+vehicle] = 5.01 ± 0.30; p < 0.01, Tukey’s post hoc). MMP-9 activity was not statistically sig-nificant among the groups on the contralateral (nonlesion) side of the brain (Fig. 3). These data suggest that MSC in-fusion might decrease the activity of MMP-9 and suppress hemorrhagic events.

Evans Blue AnalysisTo study the mechanistic insight beyond the MMP-9 ac-

tivation, the status of endothelial function 1 day after treat-ments was evaluated to analyze the EvB dye extravasation (Fig. 4). The pattern of BBB leakage evaluated with EvB in the 4 groups is similar to that of MMP-9 activity. Mac-roscopic images demonstrated intense blue EvB extravasa-




tion in the infarcted lesion on the ipsilateral side 1 day after tMCAO with treatments compared with the contralateral side (Fig. 4A–H). Intense blue in the infarcted hemisphere was observed in the following order: rtPA+vehicle (Group 2) > NS+vehicle (Group 1) > rtPA+MSCs (Group 4) > NS+MSCs (Group 3; Fig. 4A–H). Quantitative analysis of total EvB content was high in Groups 1 and 2 (NS+vehicle and rtPA+vehicle, respectively). However, when MSCs were infused with (Group 4) and without rtPA (Group 3), EvB extravasation was low (Fig. 4I). It should be noted that the rtPA+MSCs group (Group 4) had lower EvB con-tent than the rtPA+vehicle group had (Group 2; p < 0.05, Tukey’s post hoc test), and EvB content in the NS+MSCs group (Group 3) was lower than that in the NS+vehicle group (Group 1 [NS+vehicle], p < 0.05, Tukey’s post hoc test). EvB content was not statistically significant among

the groups on the contralateral (nonlesion) side of the brain (p < 0.01; Fig. 4I). Collectively with the data for MMP-9 activity, these data suggest that MSC infusion might sup-press hemorrhagic events via inhibition of endothelial dys-function.

Ischemic Lesion Volume by MRI AnalysisThe ischemic lesion volume was estimated for the 4 ex-

perimental groups using in vivo MRI (see Methods). Sam-ple images for the 4 experimental groups at 7 time points are shown in Fig. 5A. Quantification of high-intensity volume is shown in Fig. 5B. DW images were obtained at 60 minutes [row “Pre (DWI)” in Fig. 5A] and con-firmed no difference in the initial stroke volume among the groups (Group 1 [NS+vehicle] = 274 ± 12 mm3, n = 9; Group 2 [rtPA+vehicle] = 279 ± 11 mm3, n = 12; Group 3

FIG. 2. Acute hemorrhagic events after rtPA therapy with MSC infusion. A: Representative images are shown for NS+vehicle, rtPA+vehicle, NS+MSCs, and rtPA+MSCs. Low-intensity areas suggest hemorrhage (arrows). B: Incidence rate of intracerebral hemorrhage at Day 1. C: Hemorrhagic volume evaluated by T2-weighted imaging (T2WI) at Day 1. D: Infused GFP-MSCs (green) were present in the infarcted hemisphere counterstained with DAPI (blue) 1 day after tMCAO. **p < 0.01, *p < 0.05, Steel-Dwass test. Bar = 3 mm (A) and 40 μm (D).


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[NS+MSCs] = 267 ± 8 mm3, n = 11; Group 4 [rtPA+MSCs] = 276 ± 14 mm3, n = 9; F[3,37] = 0.12, ANOVA, p = 0.95). T2-weighted images were obtained from the 4 groups on Days 1, 4, 7, 14, 21, and 28 after tMCAO induction.

In all groups, estimated lesion volume gradually de-creased over the time course of 28 days after tMCAO (Fig. 5B). The MSC-treated groups displayed greater volume reduction compared with vehicle-infused groups at Days 14, 21, and 28 (p < 0.01, Tukey’s post hoc test; Fig. 4B). The lesion volume of the rtPA+MSCs group (Group 4) was 22% (Day 14), 21% (Day 21), and 25% (Day 28) of the rtPA-vehicle group (Group 2), respectively.

Although there was no statistical significance, there were trends that the reduction in lesion volume was great-est for the rtPA+MSCs group (Group 4) on Days 4, 7, 14, 21, and 28, and the ischemic lesion volume was highest for the rtPA+vehicle group (Group 2) over the time course un-til Day 28 (Fig. 5B). Collectively, these results suggest that intravenous infusion of MSCs had greater therapeutic ef-ficacy for reducing the ischemic lesion volume when rtPA therapy was performed.

Dynamic Susceptibility Contrast-Enhanced Perfusion-Weighted Image

To assess rCBF, the PWI-derived CBF maps allowed further quantitative analysis of the hemodynamic changes in the defined lesion in the ischemic brain (see Methods). The rCBF ratio between rtPA therapy groups (Groups 2 and 4) were similar, and the rCBF ratio between non-rtPA therapy groups (Groups 1 and 3) was also similar at 2 hours after reperfusion of tMCAO, although the rCBF ratio in the rtPA therapy groups were about 23% higher than in the non–rtPA therapy groups (Fig. 5C). These results suggest that the acute rCBF ratio could be upregulated to 23% by the rtPA therapy with and without receiving intravenous infusion of MSCs (F[3,10] = 8.14, p < 0.01, ANOVA).

Behavioral FunctionThe maximum velocity at which the rats could run on a

motor-driven treadmill was recorded. Before tMCAO, all rats reached a velocity of 70 m/min. Twenty-four hours af-ter tMCAO, maximum velocity on the treadmill test was at

FIG. 3. Gelatin zymography. A: Activation of MMP-9 in the contralateral (nonlesion side) was low but high in the ipsilateral (lesion side) brain for NS+vehicle and rtPA+vehicle groups (1 and 2). Activation of MMP-9 was greatly reduced in the MSC-treated groups (3 and 4) on the lesion side. B: Densitometric analysis by zymography. **p < 0.01 versus contralateral hemisphere; ††p < 0.01 versus ipsilateral Group 1, §§p < 0.01 versus ipsilateral Group 2, ANOVA, F[3,12] = 15.4, Tukey’s post hoc test.




its maximum deficit. Both the MSC-infused groups (Group 3 [NS+MSCs], n = 11 rats, and Group 4 [rtPA+MSCs], n = 9 rats) had greater maximum velocity from 4 to 28 days than non-MSC-treated controls had (Group 1 [NS+vehicle], n = 9 rats, and Group 2 [rtPA+vehicle], n = 12 rats); moreover, Group 4 attained a higher velocity than Group 3 did from Day 7 to Day 28. These results indicate that intravenous administration of MSCs improved functional outcome and that the combination of infused MSCs with rtPA thera-py could facilitate quicker recovery compared with MSC delivery alone (Group 3 [NS+MSCs]). The rtPA therapy

alone (Group 2) did not provide functional recovery com-pared with the vehicle control (Group 1) during the study period even though CBF was increased in Group 2. These data are summarized in Fig. 6.

DiscussionIn the present study, we examined both the incidence

rate of intracerebral hemorrhage and hemorrhagic volume after rtPA therapy with intravenous infusion of MSCs in the acute phase of tMCAO and demonstrated that infused

FIG. 4. EvB analysis. Extravasation of EvB (blue color) was markedly observed in the infarcted lesion 1 day after tMCAO with treatments. A–H: Macroscopic images of EvB extravasation in the infarcted lesion on the ipsilateral side. I: Quantitative analysis of total EvB content. **p < 0.01 versus contralateral hemisphere; †p < 0.05 versus ipsilateral Group 1; §p < 0.05 versus ipsilateral Group 2, ANOVA, F(3,12) = 8.3, Tukey’s post hoc test. Bars = 3 mm (A–D), 2 mm (E–H).


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MSCs reduce endothelial dysfunction. Although the rtPA therapy with vehicle infusion (without MSCs) had the highest incidence rate of intracerebral hemorrhage and greatest stroke volume, intravenous infusion of MSCs with rtPA therapy after tMCAO resulted in a greater reduction in both the incidence rate of intracerebral hemorrhage and hemorrhagic volume.

The molecular mechanisms that underlie hemorrhagic events after rtPA therapy remain unclear; however, previ-ous studies suggested the deleterious role of MMP-9 in the development of vascular damage by disrupting vascu-lar integrity during reperfusion in acute stroke.2 Ischemic insult causes an activation of MMP-9 mediated through elevation of cytokines, such as interleukin (IL)-1b and tu-mor necrosis factor (TNF)-a.35 However, it has been sug-gested that transplanted MSCs may protect damaged tis-sue by blocking TNF-a and IL-1.21 Exogenous rtPA also upregulates nuclear factor kappa light-chain enhancer of activated B cells (NF-kb) via the proteinase-activated re-

ceptor 1 pathway.24 NF-kb triggers activation of MMP-9 expression.10 Recent studies have suggested that MSCs se-crete TNF-inducible gene 6 protein, which decreased Toll-like receptor 2/NF-kb signaling through direct interaction with CD44 (for review see Prockop and Oh25). Thus, the reduction of NF-kb might inhibit activation of MMP-9.24 Taken together, the infused MSCs might protect the dis-ruption of vasculature by nondegradation of basal lamina and/or extracellular matrix with the reduced expression of MMP-9. Quantitative analysis of the EvB leakage demon-strated that dysfunction of the BBB might support the find-ings of MMP-9. Thus, it is conceivable that infused MSCs inhibit the endothelial dysfunction and may reduce BBB disruption to decrease both the incidence rate of intrace-rebral hemorrhage and hemorrhagic volume even though rtPA was infused in the acute phase of cerebral ischemia.

The rtPA-infused groups (Groups 2 and 4) showed in-creased rCBF in the lesion area 2 hours after reperfusion, and MSC-infused groups had therapeutic efficacy, which

FIG. 5. Characterization of ischemic lesion size by MRI analysis. A: Axial DW images were obtained 60 minutes after tMCAO induction (Pre [DWI]). T2-weighted images were obtained from the 4 groups at Day 1, Day 4, Day 7, Day 14, Day 21, and Day 28 after tMCAO induction from the 4 groups (NS+vehicle, rtPA+vehicle, NS+MSCs, rtPA+MSCs). Bar = 3 mm. B: A summary of lesion volume evaluated with DWI (pre) and T2-weighted imaging were obtained 1, 4, 7, 14, 21, and 28 days after reperfusion in 4 experimental groups. C: Summary of rCBF evaluated with PWI. *p < 0.05, **p < 0.01, Tukey’s post hoc tests.




is consistent with previous studies showing beneficial ef-fects of MSC infusion in experimental cerebral ischemic models.6,9,16,20 Interestingly, there was a synergistic effect of rtPA therapy and MSC infusion on ischemic stroke, in-dicated by greater functional improvement and reduced stroke volume over the study period, although the rtPA therapy alone (Group 2) did not provide therapeutic effica-cy. MSC may enhance the therapeutic efficacy contributed in part by improvement in microcirculation by rtPA. Yet, improvement in microcirculation by rtPA alone did not provide functional improvement. Thus, rtPA may improve microcirculation, allowing for greater therapeutic poten-tial of the MSCs, and the MSCs are initially important because they reduce the potential hemorrhagic complica-tions of rtPA treatment.

In the present study, we showed that there is accumu-lation of intravenously administered MSCs in the injured region in CNS diseases, including stroke, which is consis-tent with previous studies.4,16,30,34 It could be possible that infused MSCs migrated into the injured CNS tissue and provide beneficial effects. A hypothetical sequence of po-tential therapeutic mechanisms in a cell-based therapeu-tic approach for acute CNS insult is that beneficial effects may be provided from neuromodulators by MSC release, such as brain-derived neurotrophic factor15,23 at early post-MSC infusion times (days). MSCs could also support tro-phic effects for vulnerable neurons and antiinflammatory responses and the reduction of edema, thus leading to en-hanced tissue sparing following trauma.8,16,19 Over time, MSCs may contribute to vascular stabilization and remod-eling of the BBB after injury,17 thereby protecting intact tissue and reducing edema. MSCs may also induce angio-genesis,14,20,26,32,33 remyelination, and axonal sprouting.8,28,29

Cell-based therapy is considered appropriate for several

neurological diseases, including stroke.7,8 The use of rtPA is now an established stroke treatment within several hours of ischemic stroke onset. It is conceivable that patients who receive acute rtPA therapy could be infused with MSCs in a clinical setting in the future. This study might encourage this protocol, which could facilitate the therapeutic effects of MSCs in stroke.

ConclusionsSystemically administered MSCs reduced intracranial

hemorrhage by rtPA after tMCAO. The rtPA therapy also might enhance the therapeutic efficacy of MSCs.

AcknowledgmentsThis work was supported in part by the Japan Society for the

Promotion of Science KAKENHI grant nos. 26350618, 25462226, 24592138, 26462214, 15K10365, and 16K10730, and the RR&D Service of the Department of Veterans Affairs (B7335R, B9260L), the National Multiple Sclerosis Society (RG2135), and CT Stem Cell Research Program (12-SCB-Yale-05). We are thankful to the National BioResource Project for the Rat (http://www.anim.med.kyoto-u.ac.jp/NBR/) for providing this strain of rat (NBRP rat no. 0273, W-Tg [CAG-GFP] 184Ys).

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FIG. 6. Functional outcome following rtPA therapy with MSC infusion evaluated by the treadmill stress test. The maximum speed at which the rats could run on a motor-driven treadmill was recorded. An increase in maximum velocity was observed for the rtPA+MSCs group at Day 1. Both MSC-infused groups had greater maximum velocity from 7 to 28 days than non-MSC-treated groups had, but the rtPA+MSCs group at-tained a higher velocity than the NS+MSCs group from Day 7 to Day 28. *p < 0.05, **p < 0.01, Tukey’s post hoc tests.


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DisclosuresThe authors report no conflict of interest concerning the materi-als or methods used in this study or the findings specified in this paper.

Author ContributionsConception and design: M Sasaki, Nakazaki, Honmou. Acqui-sition of data: Nakazaki, T Namioka, A Namioka, Suzuki, Y Sasaki. Analysis and interpretation of data: M Sasaki, Nakazaki, Kataoka-Sasaki, Nagahama, Mikami, Wanibuchi, Kocsis, Hon-mou. Drafting the article: M Sasaki, Kocsis, Honmou. Critically revising the article: M Sasaki, Oka, Onodera, Kocsis, Honmou. Reviewed submitted version of manuscript: M Sasaki, Kataoka-Sasaki, Oka, T Namioka, A Namioka, Onodera, Suzuki, Y Sasaki, Nagahama, Mikami, Wanibuchi, Kocsis, Honmou. Approved the final version of the manuscript on behalf of all authors: M Sasaki. Statistical analysis: Onodera. Administrative/technical/material support: Kataoka-Sasaki, Suzuki, Nagahama. Study supervision: M Sasaki, Kocsis, Honmou.

CorrespondenceMasanori Sasaki, Department of Neural Regenerative Medicine, Research Institute for Frontier Medicine, Sapporo Medical Uni-versity School of Medicine, Sapporo, Hokkaido 060-8556, Japan. email: [email protected].


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Intravenous infusion of mesenchymal stem cells inhibits … · epeRfusion therapy with intravenous recombinant tissue plasminogen activator (rtPA) administra-tion is the international