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

Gila Pirzad Jahromi, Alireza P Shabanzadeh, Mina Mokhtari Hashtjini, Seyed Shahabeddin Sadr, Javad Rasouli Vani, Javad Raouf Sarshoori, Jason Charish, Gila Pirzad Jahromi, Alireza P Shabanzadeh, Mina Mokhtari Hashtjini, Seyed Shahabeddin Sadr, Javad Rasouli Vani, Javad Raouf Sarshoori, Jason Charish

Abstract

Objectives: The beneficial outcomes of bone marrow-derived mesenchymal stem cell (BMSC) treatment on functional recovery following stroke has been well established. Furthermore, 5-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase inhibitors have also been shown to increase neuronal survival and promote the movement of BMSCs towards the sites of inflammation. However, the precise mechanisms mediating the improved neurological functional recovery in stoke models following a combination treatment of Simvastatin and BMSCs still remained poorly understood.

Materials and methods: Here, an embolic stroke model was used to experimentally induce a focal ischemic brain injury by inserting a preformed clot into the middle cerebral artery (MCA). Following stroke, animals were treated either with an intraperitoneal injection of Simvastatin, an intravenous injection of 3 ×106 BMSCs, or a combination of these two treatments.

Results: Seven days after ischemia, the combination of Simvastatin and BMSCs led to a significant increase in BMSC relocation, endogenous neurogenesis, arteriogenesis and astrocyte activation while also reducing neuronal damage when compared to BMSC treatment alone (P<0.001 for all). In addition, based on western blot analysis, following stroke there was a significant decrease in c-Fos expression (P<0.001) in the combination treatment group.

Conclusion: These results further demonstrate the synergistic benefits of a combination treatment and help to improve our understanding of the underlying mechanisms mediating this beneficial effect.

Keywords: Behavioral assessment; Bone marrow stromal cell; Brain; Ischemic stroke; Simvastatin; c-Fos.

Conflict of interest statement

The authors declare that they have no conflict of interest.

Figures

Figure 1
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)
Figure 2
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)
Figure 3
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)
Figure 4
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)
Figure 5
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
Figure 6
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)
Figure 7
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)
Figure 8
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)

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