Separation And Culturing Reactive Astrocytes In Injured Adult Rats Spinal Cord And Identification Of Their Pluripotency

Objective To separate and culture the activated astrocytes in spinal cord injury andexplore the culturing system for reactive astrocytes, Also, To clarify the stem cell’scharacters of reactive astrocytes and provide the evidence for clinical therapy in spinalcord injury.Methods After the injury of spinal cord in Sprague-Dawley adult rats for1week, thespinal cord was removed to culture in DMEM/F12medium supplied with epidermalgrowth factor(EGF), basic fibroblast growth factor(bFGF) and B27; At the same time,the normal astrocytes were cultured with DMEM/F12medium contained10%Fetalbovine serum(FBS). Then the third generation of normal astrocytes was scratched by apipet to induce the reactive astrocytes produce. The BrdU was added to the culturemedium of reactive astrocytes spheres for identifying the proliferation of reactiveastrocytes. Immunocytochemistry was used to identify the differentiated cells ofreactive astrocytes to assay the marker of them. Also, Western blot was performed toevaluate the GFAP and nestin expressing in scratched cells.Results The primary culture astrocytes could adhere to flask growth and the primaryculture reactive astrocytes suspending in medium. Both reactive astrocytes separatedand cultured in vivo and induced generate in vitro were co-express GFAP and nestin byimmunocytochemistry. After induction of reactive astrocytes to differentiate, the mostdifferentiated cells from reactive astrocytes origin in vivo immediately showed GFAP+, partly MAP-2+and few RIP+, but they origin from scratched astrocytes in vitro showedGFAP+/nestin+and GFAP-/RIP-. Western blot assay the expressing of GFAP and nestinin different time stamps in scratched astrocytes. GFAP showed increase in1week andnestin emerged until1week.Conclusion Astrocytes can be activated to form reactive astrocytes in spinal cordinjury and they can co-express GFAP (the special marker of astrocytes) and nestin(special marker of neural stem cell). The results of immunocytochemistry ofdifferentiated cells indicate the reactive astrocytes emerge the characters of neural stemcell which may provide the theory for reactive astrocytes clinical therapy.