The neovascularization effect of bone marrow stromal cells in temporal muscle after encephalomyosynangiosis in chronic cerebral ischemic rats

Abstract

Objective: In Moyamoya disease, the primary goal of treatment is to improve collateral circulation through angiogenesis. In the present study, we obtained and sub-cultured bone marrow stromal cells (BMSCs) from rats without a cell-mediated immune response. Then, we injected the labeled BMSCs directly into adjacent temporal muscle during encephalomyosynangiosis (EMS). Three weeks after BMSC transplantation, we examined the survival of the cells and the extent of neovascularization.

Methods: We divided 20 rats into a BMSC transplantation group (n=12) and a control group (n=8). Seven days after the induction of chronic cerebral ischemia, an EMS operation was performed, and labeled BMSCs (1x106(6)/100 microL) were injected in the temporal muscle for the transplantation group, while an equivalent amount of culture solution was injected for the control group. Three weeks after the transplantation, temporal muscle and brain tissue were collected for histological examination and western blot analysis.

Results: The capillary/muscle ratio in the temporal muscle was increased in the BMSC transplantation group compared to the control group, showing a greater increase of angiogenesis (p<0.05). In the brain tissue, angiogenesis was not significantly different between the two groups. The injected BMSCs in the temporal muscle were vascular endothelial growth factor (VEGF)-positive by immunofluorescence staining. In both temporal muscle and brain tissue, the expression of VEGF by western blot analysis was not much different between the two groups.

Conclusion: During EMS in a chronic cerebral ischemia rat model, the injection of BMSCs resulted in accelerated angiogenesis in the temporal muscle compared to the control group.

Keywords: Angiogenesis; Bone marrow stromal cells; Chronic cerebral ischemia; Vascular endothelial growth factor.

Fig. 1

A : The encephalomyosynangiosis operation is performed in the right temporo-parietal area after 7 days of chronic cerebral ischemia. We perform a 4-mm diameter-sized craniectomy. B : Schematic drawings showing the injection sites (X).

Fig. 2

A : The injection of BMSCs into the temporal muscle increase angiogenesis. Chronic cerebral ischemia is induced by bilateral common carotid artery ligation, and BMSCs are injected into the temporal muscle 7 days after the induction. The capillary/muscle ratio is increased in the BMSCs transplantation group compared to the control group (p=0.011). B and C : Photomicroscopic examination findings of the temporal muscles stained with India ink and DAPI also shows increased angiogenesis in the BMSC transplantation group. (B) The temporal muscle of the BMSCs transplantation group shows a more prominent capillary/muscle ratio than the (C) control group. The nuclear DNA is stained with DAPI (blue dot), and the capillary is stained with India ink (black dot)(Magnification ×200). DAPI : diamino-2-phenylindole 2 HCL, BMSCs : bone marrow stromal cells.

Fig. 3

Identification of PKH26-labeled bone marrow stromal cells before injection into the temporal muscle (Magnification ×200)

Fig. 4

A : Double-labeled microphotographs for PKH26/VEGF at temporal muscle in the BMSC transplantation group. B : Nuclear DNA is stained with DAPI (blue dot) for counter staining. C : BMSCs labeled with PKH26 are visualized abundantly in the ipsilateral temporal muscle in the BMSC transplantation group. PKH26 is added to the BMSCs as a reliable marker for the identification of transplanted cells. D : Multiple VEGF-positive cells are found in the ipsilateral temporal muscle of the BMSC transplantation group. Merged image from the same view as B and C. Multiple VEGF-positive cells are co-localized with PKH26 positive cells (Magnification ×200). VEGF : vascular endothelial growth factor, DAPI : diamino-2-phenylindole 2 HCL, BMSCs : bone marrow stromal cells.

Fig. 5

The localization of transplanted cells in the temporal muscle at the chronic cerebral ischemic rat during encephalomyosynangiosis operation. A : Fluorescence-labeled BMSCs injected into the temporal muscle at 7 days after induction of ischemia are examined by microscopy at 3 weeks after the BMSCs injection. B : Without the fluorescence filter, the black dots represent vessels stained by India ink among the muscle fibers. C : With the red fluorescence filter, multiple red fluorescence-labeled cells are observed. Merged image from the same view as A and B. Fluorescence-labeled cells are usually detected around the vessels, but not directly incorporated into the vessels themselves (magnification ×200). BMSCs : bone marrow stromal cells.

Fig. 6

Double-labeled microphotographs for PKH26/VEGF at temporal muscle in the control group. A : Nuclear DNA is stained with DAPI (blue dot) for counter staining. B : Some VEGF-positive cells are found in the temporal muscle of the control group (Magnification ×200) VEGF : vascular endothelial growth factor, DAPI : diamino-2-phenylindole 2 HCL.

Fig. 7

Western blot demonstrating VEGF protein expression in the ipsilateral temporal muscle (A) and in the brain (B) after the encephalomyosynangiosis operation. A single band around 45 Kd is detected with rabbit polyclonal antibody against VEGF in the brain, and a double band is detected around 40 Kd in muscle. Both muscles and brain tissue show no definite difference in levels of expression. VEGF : vascular endothelial growth factor.