Survival and differentiation of human embryonic neural stem cells in rats with brain injury / 中国组织工程研究

ABSTRACT

BACKGROUND:Finding of neural stem cells(NSCs)brings new hope for repairing central nervous system(CNS)injury.However,the influence of internal environment after brain injury on the survival and differentiation of NSCs is a complexand variable process.OBJECTIVE:To observe the survival and differentiation of human embryonic NSCs following implantation into rats with fluid percussion brain injury.DESIGN:Open experiment.SETTING:Department of Neurosurgery,Guangdong Provincial Hospital of Traditional Chinese Medicine;Beijing Institute of Neurosurgery.MATERIALSThis experiment was carried out In the Laboratory of Neural Stem Cells,Beijing Institute of Neurosurgery from September 2002 to March 2003.Twenty-four female SD rats,aged 7 weeks,with body mass of(250±10)g,were provided by the Experimental Animal lnstitute, Chinese Academy of Medical Sciences[License Nd. SCXK(Jing)2002-2003]. Cerebrum of 8-week aborted fetus was obtained (Informed consents were obtained from parturients and their relatives). Fetal survival was monitored by B ultrasonic wave during abortion. BrdU monoclonal antibody(Sigma Company),rabbit anti-nidogen polyclonal antibody(Chemicon Company),mouse anti-microtubule-associated protein 2 (MAP-2)monoclonal antibody(Neomarkers Company),rabbit anti-gliaI fibrillary acidic protein(GFAP)polyclonal antibody (Biogenex Company).METHODS:① CerebraI cortex cells of 8-week aborted human fetus was harvested and cultured in vitro for obtaining human embryronic NSCs.②Rat models of hydraulic impact injury were developed.Bone window of motor sensory area of cerebral cortex was set at 2.5mm posterior to bregma which was zero point and 3.0 mm lateral to midline.Hydraulic impact injury parameters were set as impact pressure 0.3 MPa. impact time 25 ms and impact time once. ③BrdU-labeled human embryronic NSCs were implanted into injured area al 24 hours after injury.After 1 and 4 weeks.rats were sacrificed.Adjacent sections were doubly stained bv BrdU/MAP-2 and BrdU/GFAP.MAIN OUTCOME MEASURES:①Survival and immigration of implanted human embryonic NSCs.②Differentiation of implanted human embryonic NSCs.RESULTS:①BrdU-positive cells were oval and brown.At 1 and 4 weeks after implantation,BrdU-positive cells survived and migrated,and they migrated more widely at 4 weeks after implantation. ②At 1 week after implantation,more BrdU-positive cells were found in the subcortical granular layer and subcortex,and BrdU/MAP-2-positive cells were more than BrdU/GFAP-positive cells;At 4 weeks after implantation,BrdU-positive cells were significantly reduced,and found in choroid plexus and blood capillary,and BrdU/GFAP-positive cells were more than BrdU/MAP-2-positive cells.CONCLUSION:Implanted human embryonic NSCs can survive in the region of brain injury.gradually differentiate into astrocytes during rehabilitation and are easily digested by endothelial phagocytes.It indicates that immunological rejaction possibly influences the survival of human embryonic NSCs.