Genetically Modified Neural Stem Cell Transplantation Research On Nerve And Vascular Protective Effects After Cerebral Ischemia

Objective: To observe the survival, migration and differentiation of transplanted neural stem cells in penumbra zone of cerebral ischemia. To investigate the value of neural stem cells transplantation for recovery of injured brain. To detect gene expression of VEGF-transfected neural stem cells in transplanted area, and its effectiveness in protection of vascular and neural tissue. To discuss the possible potential with transplantation of transfected neural stem cells against cerebral ischemia.Methods: Fetal hippocampus tissues were isolated from E14 days SD rats and cultured in no-serum medium with EGF, bFGF and supplements after digestion. Single cell cloning was undertaken in order to purify the cells. pcDNA-3/VEGF121 plasmid was proliferated and the DNA sequence was checked. VEGF121 gene transfected neural stem cells with lipofectamine. Gene expression of transfected cells were detected by RT-PCR and immunofluorescent staining in vitro. Temporary middle cerebral artery occlusion (tMCAO) models were established and neurological deficit was evaluated with Neurological Severity Score (NSS). The tMCAO models were randomly divided into control group, PBS transplantation group, neural stem cells transplantation group and VEGF-secreting neural stem cells transplantation group. 3 days later, BrdU-labelled neural stem cells and VEGF-secreting neural stem cells were transplanted into the penumbra zones respectively. NSS was checked in all groups 2,4,6,8,10,12 weeks after transplantation. By using immunofluorescent and immunohistochemistry staining, VEGF expression of transplanted cells 1 week after transplantation, differentiation and migration of transplanted neural stem cells 12 week after transplantation and angiogenesis in transplanted area were detected respectively.Results: Cultured and purified neural stem cells showed nestin positive with immunofluorescent staining and had the capacity of self-renewing and multi-differentiation. VEGF121 gene transfected some of these cells with lipofectamine/plasmid complex. RT-PCR examination showed that VEGF-transfected neural stem cells could continuously express geneproducts during the first 2 weeks. Both transfected neural stem cells and their progeny expressed VEGF gene products, which was demonstrated by fluorescence study. There were no significant differences in NSS in different groups when tMCAO models were just established. However, NSS in VEGF-secreting neural stem cells transplantation group was significantly lower compared with those in other 3 groups 2-12 weeks after transplantation. NSS in neural stem cells transplantation group was also lower than those in control group and PBS transplantation group 8 and 12 weeks after transplantation. 1 week after transplantation, immunofluorescent staining showed that VEGF-transfected neural stem cells migrated and expressed VEGF in hosts' brains. 12 weeks after transplantation, immunofluorescent staining findings also suggested that transplanted neural stem cells survived and migrated, some of them differentiated to neurons and integrated well with hosts' cytoarchitectural components. Immunohistochemistry staining showed a larger amount of capillary vessel in transplanted regions in VEGF-secreting neural stem cells transplantation group.Conclusion: Neural stem cells transplantation may promote recovery of injured brain during the late period of cerebral ischemia. VEGF-transfected neural stem cells express gene products during the early time after transplantation, which reduce brain injury through protecting the vascular system against ischemia and reperfusion injury. Transplantation of VEGF-transfected neural stem cells might be a novel method for treatment of cerebral ischemia.