Culture of neural stem cells from cerebral cortex of newborn rats and its effect in cell replace in siblings / 中华围产医学杂志

ABSTRACT

Objective To isolate and culture the neural stem cells (NSCs) from the cerebral cortex of newborn rats, and investigate the cell-replace responses of the NSCs transplanted into the sibling rats with focal cerebral cortex ischemic lesion. Methods The serum-free medium DMEM/F12 (1∶1) containing basic fibroblast growth factor (bFGF or FGF2) and epidermal growth factor (EGF) was used to culture the neural stem cell spheres. The NSCs were identified by detecting the neural stem cell marker nestin with enzyme immune assay and inducing neural stem cell spheres to differentiation. The NSCs which would be used in the transplantation experiment were labeled by BrdU incorporation when cultured in vitro. The focal ischemic models were made by opening the skulls and removing the cerebral menings of 4-day-old rats to stop the blood supply for the neopallium. The BrdU-labeled NSCs were transplanted into the cerebral lesion boundary zones of the focal ischemic sibling rat models. The experiment rats were divided into lesion-transplantation group, lesion-control group and sham-operation-control group. Recipients were killed and the brains were examined by detecting the BrdU-labeled cells with enzyme immunohistochemistry at 4,7,14,30 days postgrafting, indicating the grafts living and migration in the host. Results The neural stem cell spheres, which floated and grew in medium, expressed nestin, as well as gave rise to neurons and astrocytes, could be obtained through culturing the cells derived from the cerebral cortex of newborn rats in vitro for a week. In the transplantation of the NSCs, the grafts were easy to migrate along the boundary zones of the focal ischemic lesions, and promoted the restore of the tissue structures in the damaged areas, the damage recovered well through the cell-replace responses. The BrdU-labeled positive cells in the lesion areas were full of the visual fields under microscope, the greatest density of the positive cells were focused in the granular layer of the injured cerebral cortex and not found in remote sites from the lesion. The number of BrdU-labeled cells gradually decreased in the brains of the sham-operation-control rats, only a few positive cells were found when examined at 14 days postgrafting, significantly less than that in the lesion-transplantation rats. Conclusions NSCs exist in the cerebral cortex of newborn rats. The ischemia can promote proliferation and graft of NSCs. The grafted NSCs play an important role in the recovery of focal cerebral cortex ischemic lesion.