The Effects Of Simvastatin On The Mobilization Of Endothelial Progenitor Cells And Angiogenesis Of Brain Tissue Of Rats After Traumatic Brain Injury: An Experimental Study

Objective 1.To investigate the isolation,culture,proliferation and identification ofendothelial progenitor cells(EPCs) from rat bone marrow in vitro.2.To investigatethe effect of simvastatin on the mobilization of endothelial progenitor cells(EPCs) ofrats and the proliferation,migaration and adhension of EPCs from rat bone marrowafter traumatic brain injury (TBI).3.To investigate the effect of simvastatin on theangiogenesis of brain tissue of rats and spatial learning capacity after traumatic braininjury (TBI).Methods 1.cells from rat bone marrow were isolated by Fercoll gradientcentrifugation.Isolated cells were cultured in EGM-2 medium.EPCs were identifiedby means of immunofluorescence and FACS(fluorescence activated cell sorter).Incubating EPCs with DiI in the working solution for 5 minutes at 37℃,and then foran additional 15 minutes at 4℃.Then observe the fluorescence in EPCs withfluorescence microscope.2.Adult male Wistar rats were divided into two groups:(1)saline control group (n=12);and (2) simvastatin-treated group (n=12).Fluidpercussion injury was performed over the right parietal lobe.Simvastatin wasadministered orally at a dose of 20mg/kg starting at day 1 after TBI and then daily for14 days.CD34+/CD133+ EPCs were measured by flow cytometric analysis beforeTBI and at 3,7and 14days after TBI.Mnonuclear cells from rat' bone marrow isolatedby Ficoll gradient centrifugation.Isolated cells were cultured in EGM-2 medium.EPCs were identified by means of immunofluorescence.EPCs proliferation,migaration and adhension were assayed with MTT assay,Transwell chamber assayand adhension assay.3.Adult male Wistar rats were divided into three groups:(1)sham control group (n=10);(2) saline control group (3) simvastatin-treated group(n=10).Fluid percussion injury was performed over the right parietal lobe.Simvastatin was administered orally at a dose of 20mg/kg starting at day 1 afterTBI.CD34 and CD31 were measured by immunofluorescence andimmunohistochemistry respectively.Morris water maze test was used to assess thespatial learning. Results 1.Attached cells exhibited spindled like after 4 days' culture.On the 7days,the spindled like cells increased.On the 7 days,colony were found..On the 10days,the network structure were found.More than 90% and 80% attached cells weredouble positive for Di-LDL uptake and lectin binding after 7 and 10 days' culturerespectively.FACS analysis also demonstrated that more than 34% attached cellswere double positive for CD133 and CD34.2.Simvastatin increased the mobilizationof endothelial progenitor cells(EPCs) of rats after TBI.In vitro,simvastatin strikinglyimproved the ability of proliferation,migaration and adhension of EPCs.Simvastatinat 1μmol/l concentration reached the maximum effects on EPCs.3.Treatment of TBIwith statins can increases the number ofCD34~+ cells and MVD of zone around thedamaged brain t i s sues and in the dentate gyrus of the hippocampus and improvesspatial learning on days 21-25 after onset of TBI.Conelusion 1.After induced by EGM-2 medium,EPCs can be obtained from ratbone marrow,which provides the basic for EPCs transplantations.2.Simvastatinimprove the mobilization of endothelial progenitor cells(EPCs) of rats after TBI.Inaddition,Simvastatin can improve the ability of proliferation,migaration andadhension of EPCs.3.These data suggest statin treatment augmentsTBI-induced angiogenesis,thereby leading to restoration of cognitive function afterTBI in rats.