Proliferation and differentiation of reactive nestin~+/GFAP~+ cells in an adult rat model of compression-induced spinal cord injury

摘要

BACKGROUND:Studies have demonstrated that astrocytes may possess similar properties to neural stem cells/neural precursor cells and have the potential to differentiate into neurons. OBJECTIVE:To observe neuroepithelial stem cell protein(nestin) and glial fibrillary acidic protein (GFAP) expression following spinal cord injury,and to explore whether nestin^+/GFAP^+ cells,which are detected at peak levels in gray and white matter around the ependymal region of the central canal in injured spinal cord,possess similar properties of neural stem cells. DESIGN,TIME AND SETTING:A randomized,controlled experiment.The study was performed at the Key Laboratory of Environment and Genes Related to Diseases(Xi'an Jiaotong University), Ministry of Education between January 2004 and December 2006. MATERIALS:Rabbit anti-rat nestin,β-tubulinⅢ,mouse anti-rat GFAP,galactocerebroside(GaLC) antibodies were utilized,as well as flow cytometry. METHODS:A total of 60 male,Sprague Dawley rats,aged 8 weeks,were randomly assigned to control(n = 12) and model(n = 48) groups.The spinal cord injury model was established in the model group by aneurysm clip compression,while the control animals were not treated.The gray and white matter around the ependymal region of the central canal exhibited peak expression of nestin^+/GFAP^+ cells.These cells were harvested and prepared into single cell suspension,followed by primary and passage cultures.The cells were incubated with serum-containing neural stem cell complete medium. MAIN OUTCOME MEASURES:Nestin and GFAP expression in injured spinal cord was determined using immunohistochemistry and double-labeled immunofluorescence at 1,3,5,7,14,28,and 56 days post-injury.In addition,cell proliferation and differentiation were detected using immunofluorescence cytochemistry and flow cytometry. RESULTS:Compared with the control group,the model group exhibited significantly increased nestin and GFAP expression(P<0.05),which reached peak levels between 3 and 7 days.The majority of cells in the ependymal region around the central canal were nestin^+/GFAP^- cells,while the gray and white matter around the ependymal region were full of nestin^+/GFAP^+ cells,with an astrocytic-like appearance.A large number of nestin^+/GFAP^+ cells were observed in the model group cell culture,and the cells formed clonal spheres and displayed strong nestin-positive immunofluorescence staining.Following induced differentiation,a large number of GaLC-nestin,β-tubulinⅢ-nestin,and GFAP-nestin positive cells were observed.However,no obvious changes were seen in the control group.Cells in S stage,as well as the percentage of proliferating cells,in the model group were significantly greater than in the control group(P<0.01). CONCLUSION:Spinal cord injury in the adult rat induced high expression of nestin^+/GFAP^+ in the gray and white matter around the ependymal region of the central canal.These nestin^+/GFAP^+ cells displayed the potential to self-renew and differentiate into various cells.The cells could be neural stem cells of the central nervous system.