Bone marrow-derived mesenchymal stem cell and simvastatin treatment leads to improved functional recovery and modified c-Fos expression levels in the brain following ischemic stroke.

Figures

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  Figure 1. Staining of brain sections with 5-bromodeoxyuridine (BrdU) in groups of bone marrow stromal cells (BMSC) (A) or combination treated (B). Arrows display diaminobenzidine (DAB)-labeled cells (brown). (C) Graphs of the number of BrdU-positive cell numbers in the BMSC group in comparison with the combination treated (comb) group. Results based on means±SEM. ### P<0.001 versus BMSC group (n=4 per group)

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  Figure 2. Increased arteriogenesis due to combining bone marrow stromal cells (BMSCs) and Simvastatin. Sample images of Alpha smooth muscle actin (α-SMA) immunostaining following middle cerebral artery occlusion (MCAO) from control (A), BMSC-treated (B), Simvastatin treated (C) and combination of Simvastatin and BMSC (D) groups. Arrows indicate diaminobenzidine (DAB)-labeled α-SMA-vessels (brown). (E) Arterial density quantification of control (cont), BMSC- treated (BMSC), Simvastatin- treated (sim), and combination-treated (comb) groups. Results based on means±SEM. * P<0.05, *** P<0.001 versus control group, ++ P<0.01 compare with Simvastatin group and ## P<0.01 compare with BMSC group (n = 4 per group)

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  Figure 3. Neurological behavior after ischemic stroke. Neurological scores following stroke, based on the modified Bederson scoring system, for experimental groups (A). Adhesive removal test times at 24 hr and 7 days following stroke for experimental groups (B). Results are based on means ± SEM. * P<0.01, ** P<0.01, ***P<0.001 compare to control group, + P<0.05, ++P<0. 01, +++ P<0.001 compare to Simvastatin group, # P<0.5, ## P <0.01, ### P<0.001 compare to bone marrow stromal cells (BMSC) group (n = 8 per group)

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  Figure 4. (A) Representative images of hematoxylin and eosin–stained coronal sections 7 days post- middle cerebral artery occlusion (MCAO) illustrating ischemic areas in experimental groups. (B) Quantification of infarct volume in the various treatment groups. Results are based on means±SEM. *** P<0.001 vs control, ++ P<0.05 compare to Simvastatin. # P<0.05 vs bone marrow stromal cells (BMSC) (n = 8 per group)

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  Figure 5. Increased astrocyte density following treatment with bone marrow stromal cells (BMSCs) and Simvastatin. Sample images of glial fibrillary acidic protein (GFAP) immunostaining in control middle cerebral artery occlusion (MCAO) rats (A), BMSC-treated (B), Simvastatin-treated (C) and combination-treated (D) rats.(E) quantifications of A-D (n = 4 per group). Results are based on mean±SEM. *** P<0.001 versus control rats, +++ P<0.001 compared to rats treated with Simvastatin and ## P<0.01 versus rats treated with BMSC. Arrows indicate diaminobenzidine (DAB)-labeled astrocytes (brown) located in the injury border zone. Asterisks indicate the infarcted zone at 7 days after MCAO

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  Figure 6. Increased numbers of proliferating cell in the subventricular zone (SVZ) of the ischemic brain following bone marrow stromal cells (BMSC) and Simvastatin combination treatment. Staining for Ki-67 in control (A), BMSC (B), Simvastatin (C) and combination (D) treated rats following middle cerebral artery occlusion (MCAO). (E) Quantification of data in A-D. Results are based on mean±SEM. * P<0.05 vs MCAO controls, *** P<0.001 vs MCAO controls, +++ P<0.001 vs Simvastatin group and ### P<0.01 vs BMSC group. Arrows indicate diaminobenzidine (DAB)-labeled proliferating cells (brown). The SVZ of the lateral ventricle in the injured hemisphere at 7 days after MCAO is shown with asterisks. (n= 4 per group)

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  Figure 7. Decreased neuronal destruction following bone marrow stromal cells (BMSC) and Simvastatin combination treatment in middle cerebral artery occlusion (MCAO) rats. Neuron-specific enolase (NSE) staining in control (A), Simvastatin (B), BMSC (C) or combination-treated (D) MCAO rat groups. Arrows indicate diaminobenzidine (DAB)-labeled neurons (brown). (E) Plot of neuronal damage in the various treatment groups. Results are based on means ± SEM. *** P<0.001 vs control group, ++ P< 0.01 vs simvastatin group and # P<0.05 vs BMSC group (n=4 per group)

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  Figure 8. Decreased expression of c-Fos in the peri-infarct tissue in the bone marrow stromal cells (BMSC) and Simvastatin combination treatment groups following stroke. (A) Western blot analysis of brain samples for control (cont), Simvastatin (sim), BMSC or combination-treated rats (comb). (B) Quantification of expression level of c-Fos at 48 hr after embolization for the previously mentioned groups. For each sample, band intensity was normalized to β -actin. For each group data are presented as the mean of four separate brains±SEM. *** P<0.001 vs control, ++ P<0.01 vs Simvastatin and # P<0.05 vs BMSC (n=4 per group)