I.V. infusion of brain-derived neurotrophic factor gene-modified human mesenchymal stem cells protects against injury in a cerebral ischemia model in adult rat

Abstract

I.V. delivery of mesenchymal stem cells prepared from adult bone marrow reduces infarction size and ameliorates functional deficits in rat cerebral ischemia models. Administration of the brain-derived neurotrophic factor to the infarction site has also been demonstrated to be neuroprotective. To test the hypothesis that brain-derived neurotrophic factor contributes to the therapeutic benefits of mesenchymal stem cell delivery, we compared the efficacy of systemic delivery of human mesenchymal stem cells and human mesenchymal stem cells transfected with a fiber-mutant F/RGD adenovirus vector with a brain-derived neurotrophic factor gene (brain-derived neurotrophic factor-human mesenchymal stem cells). A permanent middle cerebral artery occlusion was induced by intraluminal vascular occlusion with a microfilament. Human mesenchymal stem cells and brain-derived neurotrophic factor-human mesenchymal stem cells were i.v. injected into the rats 6 h after middle cerebral artery occlusion. Lesion size was assessed at 6 h, 1, 3 and 7 days using MR imaging, and histological methods. Functional outcome was assessed using the treadmill stress test. Both human mesenchymal stem cells and brain-derived neurotrophic factor-human mesenchymal stem cells reduced lesion volume and elicited functional improvement compared with the control sham group, but the effect was greater in the brain-derived neurotrophic factor-human mesenchymal stem cell group. ELISA analysis of the infarcted hemisphere revealed an increase in brain-derived neurotrophic factor in the human mesenchymal stem cell groups, but a greater increase in the brain-derived neurotrophic factor-human mesenchymal stem cell group. These data support the hypothesis that brain-derived neurotrophic factor contributes to neuroprotection in cerebral ischemia and cellular delivery of brain-derived neurotrophic factor can be achieved by i.v. delivery of human mesenchymal stem cells.

Fig. 1

May-Giemsa staining of primary hMSCs (A) and BDNF-hMSCs (B) (scale bar=20 μm). (C) Secreted BDNF levels in supernatant of hMSCs (ng/10⁵ cells/48 h) transfected with AxCAhBDNF-F/RGD (BDNF-hMSC) at an MOI of 300, 1000, and 3000 pu/cell.

Fig. 2

Evaluation of the ischemic lesion volume with MRI T₂-weighted images. hMSCs or BDNF-hMSCs were i.v. injected immediately after the initial MRI scanning (6 h). Images obtained 6, 24, 72 h and one week after MCAO in non-treated (A1–4), hMSC-treated (B1–4), and BDNF-hMSC-treated groups (C1–4). Scale bar=3 mm.

Fig. 3

(A) Summary of lesion volumes evaluated with MRI T₂-weighted images in each group. (B) BDNF in vivo levels assayed with ELISA. Levels of BDNF were significantly increased in the ischemic hemisphere of hMSC and BDNF-hMSC compared with non-treated rats. In addition, BDNF levels were greater in the ischemic hemisphere of BDNF-hMSC-transplanted compared with rats that received hMSC. Assays were obtained 7 days after MCAO induction. (* P<0.05; ** P<0.01, *** P<0.0005.)

Fig. 4

(A–C) Brain slices stained with TTC to visualize lesions. TTC-stained brain slices from non-treated (A) and following i.v. delivery hMSCs or BDNF-hMSCs 6 h after MCAO are shown in B and C, respectively. (Scale bar=3 mm.) (D) Lesion volumes in each group. (* P<0.05; ** P<0.0005.)

Fig. 5

I.v.-administrated BDNF-hMSCs accumulated in the ischemic lesion hemisphere. BDNF-hMSCs were transfected with the reporter gene LacZ. (B) Transplanted LacZ-positive cells (blue cells) were present in the ischemic lesion. Brain from control (BDNF-hMSCs without LacZ transfection) injected animals with comparable X-gal staining is shown in A. Confocal images (white square in panel B) at low (C) and high power (D) demonstrating a large number of LacZ-positive cells in the lesion hemisphere. Scale bar=3 mm (A, B), 100 μm (C), 10 μm (D).

Fig. 6

The treadmill stress test demonstrates that the maximum speed at which the rats could run on a motor driven treadmill was faster in the hMSC-and the BDNF-hMSC-treated rats than control. Moreover, the greatest velocity was achieved in the BDNF-hMSC group. Velocity is plotted for three times after MCAO induction. (* P<0.001.)