Synergistic therapeutic effects of intracerebral transplantation of human modified bone marrow-derived stromal cells (SB623) and voluntary exercise with running wheel in a rat model of ischemic stroke

Abstract

Background: Mesenchymal stromal cell (MSC) transplantation therapy is a promising therapy for stroke patients. In parallel, rehabilitation with physical exercise could ameliorate stroke-induced neurological impairment. In this study, we aimed to clarify whether combination therapy of intracerebral transplantation of human modified bone marrow-derived MSCs, SB623 cells, and voluntary exercise with running wheel (RW) could exert synergistic therapeutic effects on a rat model of ischemic stroke.

Methods: Wistar rats received right transient middle cerebral artery occlusion (MCAO). Voluntary exercise (Ex) groups were trained in a cage with RW from day 7 before MCAO. SB623 cells (4.0 × 10⁵ cells/5 μl) were stereotactically injected into the right striatum at day 1 after MCAO. Behavioral tests were performed at day 1, 7, and 14 after MCAO using the modified Neurological Severity Score (mNSS) and cylinder test. Rats were euthanized at day 15 after MCAO for mRNA level evaluation of ischemic infarct area, endogenous neurogenesis, angiogenesis, and expression of brain-derived neurotrophic factor (BDNF) and vascular endothelial growth factor (VEGF). The rats were randomly assigned to one of the four groups: vehicle, Ex, SB623, and SB623 + Ex groups.

Results: SB623 + Ex group achieved significant neurological recovery in mNSS compared to the vehicle group (p < 0.05). The cerebral infarct area of SB623 + Ex group was significantly decreased compared to those in all other groups (p < 0.05). The number of BrdU/Doublecortin (Dcx) double-positive cells in the subventricular zone (SVZ) and the dentate gyrus (DG), the laminin-positive area in the ischemic boundary zone (IBZ), and the mRNA level of BDNF and VEGF in SB623 + Ex group were significantly increased compared to those in all other groups (p < 0.05).

Conclusions: This study suggests that combination therapy of intracerebral transplantation SB623 cells and voluntary exercise with RW achieves robust neurological recovery and synergistically promotes endogenous neurogenesis and angiogenesis after cerebral ischemia, possibly through a mechanism involving the up-regulation of BDNF and VEGF.

Keywords: Cerebral ischemic infarct; Regenerative medicine; Rehabilitation; SB623; Voluntary exercise.

Fig. 1

Experimental protocol of this study and illustration of ischemic lesion. a Study design for vehicle group (n = 9) and SB623 group (n = 9). b Study design for Ex group (n = 8) and SB623 + Ex group (n = 9). The Ex group and the SB623 + Ex group trained in a cage with RW from 7 days before MCAO and continued for 21 days. All rats were performed right MCAO. At day 1 after MCAO, all rats which were diagnosed with ischemic stroke by behavioral test were stereotactically transplanted with DMEM or SB623 cells into the right striatum. At day 15 after MCAO, all rats were euthanized, followed by histological analysis and assessment of the mRNA expression of BDNF and VEGF in the IBZ via quantitative reverse transcription polymerase chain reaction analysis. c A coronal section identified at the level of 0.3 mm posterior from the bregma in rat brain that divides the right ischemic hemisphere into two subregions (ischemic core [IC]; ischemic boundary zone [IBZ]). Abbreviation: RW: running wheel; MCAO: middle cerebral artery occlusion; DMEM: Dulbecco’s modified Eagle’s medium; BDNF: brain-derived neurotrophic factor; VEGF: vascular endothelial growth factor, IBZ: ischemic boundary zone

Fig. 2

Running wheel used in this study. The Ex group and the SB623 + Ex group were individually housed in cages with RW for 7 days before MCAO and freely exercised until day 14 after MCAO. Abbreviation: RW: running wheel; MCAO: middle cerebral artery occlusion

Fig. 3

Body weight and behavioral tests for each group. a There was a significant difference in weight gain between the vehicle group and the voluntary exercise group from day − 7 to day 0 after MCAO. Comparing day 0 and day 1 after MCAO, body weight was decreased in all groups at day 1. ANOVA revealed a significant treatment effect with significant difference in weight loss between the voluntary exercise group (Ex and SB623 + Ex groups) and the no-exercise group (vehicle and SB623 groups) at day 1 after MCAO (F_{(3, 31)} = 8.7, p < 0.001 at day 1) (mean ± SE, ^*p < 0.05 vs. vehicle group). Body weight decreased from day 1 to day 7, and increased from day 8 to day 14 in all groups. b ANOVA revealed significant treatment effects, of the Ex, SB623, and SB623 + Ex groups, respectively, in mNSS at later time points after MCAO, which significantly improved compared to that of vehicle group at day 7 and day 14 after MCAO (F_{(3, 31)} = 7.07, p < 0.001 at day 7, F_{(3, 31)} = 10.80, p < 0.001 at day 14) (mean ± SE, ^*p < 0.05 vs. vehicle group). c The graph shows the results of contralateral bias in the cylinder test (vehicle group: n = 6; Ex group: n = 7; SB623 group: n = 5; SB623 + Ex group: n = 8). ANOVA revealed that there were no significant treatment effects in cylinder test from all four groups (F_{(3, 22)} = 1.42, p = 0.26). Abbreviation: MCAO: middle cerebral artery occlusion

Fig. 4

Daily running distance with running wheel after MCAO. The running distance increased day by day between the Ex group and the SB623 + Ex groups. There was no significant difference in the average running distance from day 1 to day 14 after MCAO (mean ± SD, p = 0.58). There was no significant difference in the average running distance at day 1, day 7, and day 14 after MCAO, respectively (mean ± SD, day 1: p = 0.53; day 7: p = 0.79; day 14: p = 0.48, respectively). The highest average running distance in the Ex group was 2711 m at day 11 after MCAO (mean ± SD, p = 0.47) and 3418 m in the SB623 + Ex group at day 13 after MCAO (mean ± SD, p = 0.23). Abbreviation: RW: running wheel; MCAO: middle cerebral artery occlusion

Fig. 5

Nissl staining for cerebral infarct area and TTC staining for ischemic brain tissue in each group. a, b Cerebral infarct area is shown by Nissl staining. A a is representative Nissl staining for cerebral infarction area. The tract of red dotted line revealed cerebral infarct area. We calculated the cerebral infarct area ratio by the following method: Cerebral infarct area ratio = [LT – (RT – RI)] × 100/LT (%), where LT is the area of the left hemisphere, RT is the area of the right hemisphere, and RI is the infarct area in mm² at the level of SVZ. c ANOVA detected significant treatment effects on cerebral infarct in Nissl-stained infarcted areas, which was significantly reduced in the SB623 + Ex group compared to the vehicle, Ex, and SB623 group, respectively (each group: n = 5, F_{(3, 16)} = 16.4, p < 0.001). The Ex group and the SB623 group showed significantly reduced infarcted areas in Nissl-stained tissue sections compared to the vehicle group (mean ± SE, ^*p < 0.05, ^**p < 0.01 vs. vehicle group, ^#p < 0.05 vs. SB623 + Ex group). d Representative TTC-stained coronal sections at the level of 0.7 mm anterior from bregma are shown in each group. Infarct area appears in white, and normal tissue in red. Abbreviation: TTC: 2, 3, 4-triphenyltetrazolium chloride, MCAO: middle cerebral artery occlusion; SVZ: subventricular zone

Fig. 6

Immunofluorescent staining for endogenous neurogenesis in SVZ and DG. a, b Immunofluorescent staining for BrdU, Dcx, and DAPI shows the endogenous neurogenesis in SVZ and DG. SVZ: Scale bar = 100 μm. DG: Scale bar = 200 μm. c High-magnification photographs of a representative immunofluorescent staining for BrdU/Dcx double-positive cells in the SVZ (yellow arrows). Scale bar = 100 μm. d, e ANOVA detected significant treatment effects on endogenous neurogenesis with significantly increased numbers of BrdU/Dcx double-positive cells in SVZ and DG in the SB623 + Ex group compared to the vehicle, Ex, and SB623 groups, respectively (each group: n = 5, SVZ: F_{(3, 16)} = 18.9, p < 0.001; F_{(3, 16)} = 13.7, DG: p < 0.001). The Ex group and the SB623 group had significantly increased numbers of BrdU/Dcx double-positive cells in SVZ and DG compared to the vehicle group (SVZ: mean ± SE, ^*p < 0.05, ^**p < 0.01 vs. vehicle group, ^#p < 0.05 vs. SB623 + Ex group; DG: ^*p < 0.05, ^**p < 0.01 vs. vehicle group, ^#p < 0.05 vs. SB623 + Ex group). Abbreviation: SVZ: subventricular zone; DG: dentate gyrus; BrdU: 5-bromo2′-deoxyuridine, Dcx: Doublecortin, DAPI: 4′ 6-Diamidino-2-phenylindole

Fig. 7

Immunofluorescent staining for angiogenesis in the IBZ. a Immunofluorescent staining for the laminin-positive area to evaluate angiogenesis in the IBZ. Scale bar = 100 μm. b ANOVA detected significant treatment effects on angiogenesis as assessed by measuring the percentage of laminin-positive area, which significantly increased in the SB623 + Ex group compared to the vehicle, Ex, and SB623 groups, respectively (each group: n = 5, F_{(3, 16)} = 14.0, p < 0.001). The Ex group and the SB623 group had significantly increased in laminin-positive percentage areas in the IBZ compared to the vehicle group (mean ± SE, ^*p < 0.05, ^**p < 0.01 vs. vehicle group, ^#p < 0.05 vs. SB623 + Ex group). Abbreviation: IBZ: ischemic boundary zone

Fig. 8

Immunofluorescent staining for the viability of SB623 cells in the healthy rat after intracerebral transplantation. a Immunofluorescent staining for STEM121/DAPI double-positive cells in the healthy rat shows SB623 cells at day 3 after intracerebral transplantation (white arrow) (top: low magnification, bottom: high magnification). Scale bar = 100 μm. b ANOVA revealed that the number of STEM121-positive cells at day 3 after transplantation was significantly higher than that at day 7 and day 14 (each group: n = 6) (F_{(2, 15)} = 264, p < 0.001) (mean ± SE, ^**p < 0.01 vs. day 3 group, ^#p < 0.05 vs. day 14 group). Abbreviation: DAPI: 4′ 6-diamidino-2-phenylindole

Fig. 9

mRNA expression of BDNF and VEGF in the IBZ. ANOVA detected significant treatment effects on mRNA expressions of BDNF and VEGF, which were significantly up-regulated in the SB623 + Ex group compared to the vehicle, Ex, and SB623 groups, respectively (each group: n = 6, BDNF: F_{(3, 20)} = 10.1, p < 0.001, VEGF: F_{(3, 20)} = 13.1, p < 0.001) (mean ± SE, BDNF: F_{(3, 20)} = 10.1, p < 0.001, VEGF: F_{(3, 20)} = 13.1, p < 0.001). The Ex group and the SB623 group significantly up-regulated mRNA expressions of BDNF and VEGF compared to the vehicle group (^*p < 0.05, ^**p < 0.01 vs. vehicle group, ^#p < 0.05 vs. SB623 + Ex group). Abbreviation: BDNF: brain-derived neurotrophic factor, VEGF: vascular endothelial growth factor, IBZ: ischemic boundary zone

Fig. 10

Correlational analyses between the endogenous neurogenesis, cerebral infarct area ratio, and running distance. a, b The number of BrdU/Dcx double-positive cells in the SVZ and the DG from all four groups negatively correlated with cerebral infarct area ratio (SVZ: r = − 0.77, p < 0.01; DG: r = − 0.73, p < 0.01). c, d The number of BrdU/Dcx double-positive cells and running distance in the Ex group and the SB623 + Ex group (SVZ: r = 0.19, p > 0.05; DG: r = 2.06 p > 0.05). e There was no significant correlation between cerebral infarct area ratio and running distance (r = − 0.29, p > 0.05). Abbreviation: SVZ: subventricular zone; DG: dentate gyrus; BrdU: 5-bromo2’-deoxyuridine, Dcx: Doublecortin